Feedback control in microfluidic systems

ABSTRACT

Systems and methods for controlling fluids in microfluidic systems are generally described. In some embodiments, control of fluids involves the use of feedback from one or more processes or events taking place in the microfluidic system. For instance, a detector may detect one or more fluids at a measurement zone of a microfluidic system and one or more signals, or a pattern of signals, may be generated corresponding to the fluid(s). In some cases, the signal or pattern of signals may correspond to an intensity, a duration, a position in time relative to a second position in time or relative to another process, and/or an average time period between events. Using this data, a control system may determine whether to modulate subsequent fluid flow in the microfluidic system. In some embodiments, these and other methods can be used to conduct quality control to determine abnormalities in operation of the microfluidic system.

RELATED APPLICATIONS

The present application claims priority to U.S. provisional applicationsU.S. Ser. No. 61/325,023, filed Apr. 16, 2010, U.S. Ser. No. 61/325,044,filed Apr. 16, 2010, and U.S. Ser. No. 61/363,002, filed Jul. 9, 2010,each of which is incorporated herein by reference in its entirety.

FIELD

Systems and methods for controlling fluids in microfluidic systems aregenerally described. In some embodiments, control of fluids involves theuse of feedback from one or more processes or events taking place in themicrofluidic system.

BACKGROUND

The manipulation of fluids plays an important role in fields such aschemistry, microbiology and biochemistry. These fluids may includeliquids or gases and may provide reagents, solvents, reactants, orrinses to chemical or biological processes. While various microfluidicmethods and devices, such as microfluidic assays, can provideinexpensive, sensitive and accurate analytical platforms, fluidmanipulations—such as the mixture of multiple fluids, sampleintroduction, introduction of reagents, storage of reagents, separationof fluids, collection of waste, extraction of fluids for off-chipanalysis, and transfer of fluids from one chip to the next—can add alevel of cost and sophistication. Accordingly, advances in the fieldthat could reduce costs, simplify use, provide quality control of theanalysis being performed, and/or improve fluid manipulations inmicrofluidic systems would be beneficial.

SUMMARY

Systems and methods for controlling fluids in microfluidic systems aregenerally described. In some embodiments, control of fluids involves theuse of feedback from one or more processes or events taking place in themicrofluidic system. The subject matter of the present inventioninvolves, in some cases, interrelated products, alternative solutions toa particular problem, and/or a plurality of different uses of one ormore systems and/or articles.

In one set of embodiments, a series of methods are provided. In oneembodiment, a method comprises initiating detection of fluids at a firstmeasurement zone of a microfluidic system. The method involves detectinga first fluid and a second fluid at the first measurement zone andforming a first signal corresponding to the first fluid and a secondsignal corresponding to the second fluid. A first pattern of signals istransmitted to a control system, the first pattern of signals comprisingat least two of: a) an intensity of the first signal; b) a duration ofthe first signal; c) a position of the first signal in time relative toa second position in time; and d) an average time period between thefirst and second signals. The method also involves determining whetherto modulate fluid flow in the microfluidic system based at least in parton the first pattern of signals.

In another embodiment, a method comprises detecting a first fluid and asecond fluid at a first measurement zone of a microfluidic system,wherein the detection step comprises detecting at least two of a) anopacity of the first fluid; b) a volume of the first fluid; c) a flowrate of the first fluid; d) a position of the detection of the firstfluid in time relative to a second position in time; and e) an averagetime period between the detection of the first and second fluids. Themethod involves determining whether to modulate fluid flow in themicrofluidic system based at least in part on the detection step.

In another embodiment, a method of conducting quality control todetermine abnormalities in operation of a microfluidic system comprisesdetecting a first fluid at a first measurement zone of the microfluidicsystem and forming a first signal corresponding to the first fluid. Themethod also involves transmitting the first signal to a control system,comparing the first signal to a reference signal, thereby determiningthe presence of abnormalities in operation of the microfluidic system,and determining whether to stop an analysis being conducted in themicrofluidic system based at least in part on results of the comparingstep.

Other advantages and novel features of the present invention will becomeapparent from the following detailed description of various non-limitingembodiments of the invention when considered in conjunction with theaccompanying figures. In cases where the present specification and adocument incorporated by reference include conflicting and/orinconsistent disclosure, the present specification shall control. If twoor more documents incorporated by reference include conflicting and/orinconsistent disclosure with respect to each other, then the documenthaving the later effective date shall control.

BRIEF DESCRIPTION OF THE DRAWINGS

Non-limiting embodiments of the present invention will be described byway of example with reference to the accompanying figures, which areschematic and are not intended to be drawn to scale. In the figures,each identical or nearly identical component illustrated is typicallyrepresented by a single numeral. For purposes of clarity, not everycomponent is labeled in every figure, nor is every component of eachembodiment of the invention shown where illustration is not necessary toallow those of ordinary skill in the art to understand the invention. Inthe figures:

FIG. 1 is a block diagram showing a microfluidic system and a variety ofcomponents that may be part of a sample analyzer according to oneembodiment;

FIG. 2 is a plot showing measurement of optical density as a function oftime according to one embodiment;

FIG. 3 is a perspective view of a cassette including a fluidic connectoraccording to one embodiment;

FIG. 4 is a an exploded assembly view of a cassette according to oneembodiment;

FIG. 5 is a schematic view of a cassette according to one embodiment;

FIG. 6 is a diagram showing a microfluidic system of a cassetteincluding a fluidic connector according to one embodiment;

FIG. 7 is a schematic view of a portion of a sample analyzer accordingto one embodiment;

FIG. 8 is a block diagram showing a control system of a sample analyzerassociated with a variety of different components according to oneembodiment;

FIG. 9 is a schematic diagram showing a microfluidic system of acassette according to one embodiment; and

FIG. 10 is a plot showing measurement of optical density as a functionof time according to one embodiment.

DETAILED DESCRIPTION

Systems and methods for controlling fluids in microfluidic systems aregenerally described. In some embodiments, control of fluids involves theuse of feedback from one or more processes or events taking place in themicrofluidic system. For instance, a detector may detect one or morefluids passing across a measurement zone of a microfluidic system andone or more signals, or a pattern of signals, may be generatedcorresponding to the fluid(s). In some cases, the signal or pattern ofsignals may correspond to an intensity (e.g., an indication of the typeof fluid passing across the detector), a duration (e.g., an indicationof the volume and/or flow rate of fluid), a position in time relative toanother position in time or relative to another process that hasoccurred in the microfluidic system (e.g., when a certain fluid passesacross the detector after a valve has been actuated), and/or an averagetime period between events (e.g., between two consecutive signals).Using this data, a control system may determine whether to modulatesubsequent fluid flow in the microfluidic system. In some embodiments,these and other methods can be used to conduct quality control todetermine abnormalities in operation of the microfluidic system.

As described in more detail below, in some embodiments an analysisperformed in a device can be recorded to produce essentially a“fingerprint” of the analysis, and all or potions of the fingerprint maybe used to provide feedback to the microfluidic system. For example, afingerprint of an analysis may include signals from each fluid at (e.g.,passing across, through, above, below, etc.) a detector or multipledetectors, which may be statically positioned at a measurement zone orat multiple measurement zones of a device. The signals may be ameasurement of, for example, the transmission of light passing throughthe fluids. Since different fluids used in the analysis may havedifferent volumes, flow rates, compositions, and other characteristics,the fluids may produce signals having different intensities anddurations, which are reflected in the fingerprint. As such, thefingerprint can be used to identify, for example, the fluids used in theanalysis, the timing of the fluids (e.g., when particular fluids wereintroduced into certain regions of the device), and the interactionbetween the fluids (e.g., mixing). This data can be used to providefeedback to modulate subsequent fluid flow in the microfluidic system,and in some cases, to conduct quality control to determine whether allor portions of the analysis was run properly.

The systems and methods described herein may find application in avariety of fields. In some cases, the systems and methods may be used toconduct quality control to determine, for example, a correct sequence ofevents taking place in the microfluidic system. If an incorrect sequenceof events is determined, the feedback control may, for example, cancelthe test being performed in the microfluidic system and/or alert theuser of the abnormality. Additionally and/or alternatively, the systemsand methods described herein may be used to modulate fluid flow such asmixing, introduction or removal of fluids into certain channels orreservoirs in the microfluidic system, actuation of one or morecomponents such as a valve, pump, vacuum, or heater, and otherprocesses. These and other processes may be applied to a variety ofmicrofluidic systems such as, for example, microfluidic point-of-carediagnostic platforms, microfluidic laboratory chemical analysis systems,high-throughput detection systems, fluidic control systems in cellcultures or bio-reactors, among others. The articles, systems, andmethods described herein may be particularly useful, in some cases,where an inexpensive, robust, disposable microfluidic device is desired.

Furthermore, the feedback control described herein may be used toperform any suitable process in a microfluidic system, such as achemical and/or biological reaction. As a specific example, the feedbackcontrol may be used to control reagent transport in antibody assays thatemploy unstable reaction precursors, such as the silver solution assaydescribed in the Examples section. Other advantages are described inmore detail below.

The articles, components, systems, and methods described herein may becombined with those described in International Patent Publication No.WO2005/066613 (International Patent Application Serial No.PCT/US2004/043585), filed Dec. 20, 2004 and entitled “Assay Device andMethod”; International Patent Publication No. WO2005/072858(International Patent Application Serial No. PCT/US2005/003514), filedJan. 26, 2005 and entitled “Fluid Delivery System and Method”;International Patent Publication No. WO2006/113727 (International PatentApplication Serial No. PCT/US06/14583), filed Apr. 19, 2006 and entitled“Fluidic Structures Including Meandering and Wide Channels”; U.S. patentapplication Ser. No. 12/113,503, published as U.S. Patent PublicationNo. 2008/0273918, filed May 1, 2008 and entitled “Fluidic Connectors andMicrofluidic Systems”; U.S. patent application Ser. No. 12/196,392,published as U.S. Patent Publication No. 2009/0075390, filed Aug. 22,2008, entitled “Liquid containment for integrated assays”; U.S. patentapplication Ser. No. 12/428,372, filed Apr. 22, 2009, published as U.S.Patent Publication No. 2009/0266421, entitled “Flow Control inMicrofluidic Systems”; U.S. patent application Ser. No. 12/640,420,filed Dec. 17, 2009, entitled, “Reagent Storage in Microfluidic Systemsand Related Articles and Methods”; U.S. patent application Ser. No.12/698,451, filed Feb. 2, 2010, entitled, “Structures for ControllingLight Interaction with Microfluidic Devices”; U.S. Patent Apl. Ser. No.61/263,981, filed Nov. 14, 2009 and entitled, “Fluid Mixing and Deliveryin Microfluidic Systems; U.S. Provisional Patent Application No.61/325,044, filed Apr. 16, 2010 and entitled, “System for Analysis ofSamples”, each of which is incorporated herein by reference in itsentirety for all purposes.

A series of exemplary systems and methods are now described.

FIG. 1 shows a block diagram 10 of a microfluidic system and variouscomponents that may provide feedback control according to one set ofembodiments. The microfluidic system may include, for example, a deviceor cassette 20 operatively associated with one or more components suchas a fluid flow source 40 such as a pump (e.g., for introducing one ormore fluids into the device and/or for controlling the rates of fluidflow), optionally a fluid flow source 40 such as a pump or vacuum thatmay be configured to apply either of both of a positive pressure orvacuum (e.g., for moving/removing one or more fluids within/from thecassette and/or for controlling the rates of fluid flow), a valvingsystem 28 (e.g., for actuating one or more valves), a detection system34 (e.g., for detecting one or more fluids and/or processes), and/or atemperature regulating system 41 (e.g., to heat and/or cool one or moreregions of the device). The components may be external or internal tothe microfluidic device, and may optionally include one or moreprocessors for controlling the component or system of components. Incertain embodiments, one or more such components and/or processors areassociated with a sample analyzer 47 configured to process and/oranalyze a sample contained in the microfluidic system. Non-limitingexamples of analyzers that may be used with microfluidic systems anddevices described herein are described in more detail below and in U.S.Provisional Patent Application No. 61/325,044, filed Apr. 16, 2010 andentitled, “System for Analysis of Samples”, which is incorporated hereinby reference in its entirety for all purposes.

In general, as used herein, a component that is “operatively associatedwith” one or more other components indicates that such components aredirectly connected to each other, in direct physical contact with eachother without being connected or attached to each other, or are notdirectly connected to each other or in contact with each other, but aremechanically, electrically (including via electromagnetic signalstransmitted through space), or fluidically interconnected (e.g., viachannels such as tubing) so as to cause or enable the components soassociated to perform their intended functionality.

The components shown illustratively in FIG. 1, as well as other optionalcomponents, may be operatively associated with a control system 50. Insome embodiments, the control system may be used to control fluidsand/or conduct quality control by the use of feedback from one or moreevents taking place in the microfluidic system. For instance, thecontrol system may be configured to receive input signals from the oneor more components, to calculate and/or control various parameters, tocompare one or more signals or a pattern of signals with signals orvalues pre-programmed into the control system, and/or to send signals toone or more components to modulate fluid flow and/or control operationof the microfluidic system. Specific examples of feedback control areprovided below.

The control system may also be optionally associated with othercomponents such as a user interface 54, an identification system 56, anexternal communication unit 58 (e.g., a USB), and/or other components,as described in more detail below.

Microfluidic device (e.g., cassette) 20 may have any suitableconfiguration of channels and/or components for performing a desiredanalysis. In one set of embodiments, microfluidic device 20 containsstored reagents that can be used for performing a chemical and/orbiological reaction (e.g., an immunoassay). The microfluidic device mayinclude, for example, an optional reagent inlet 62 in fluidcommunication with an optional reagent storage area 64. The storage areamay include, for example, one or more channels and/or reservoirs thatmay, in some embodiments, be partially or completely filled with fluids(e.g., liquids and gases, including immiscible reagents such as reagentsolutions and wash solutions, optionally separated by immiscible fluids,as described in more detail below). The device may also include anoptional sample or reagent loading area 66, such as a fluidic connectorthat can be used to connect reagent storage area 64 to an optionalmeasurement zone 68 (e.g., a reaction area). Examples of fluidicconnectors are described in more detail in U.S. patent application Ser.No. 12/113,503, published as U.S. Patent Publication No. 2008/0273918,filed May 1, 2008 and entitled “Fluidic Connectors and MicrofluidicSystems”, which is incorporated herein by reference in its entirety. Themeasurement zone, which may include one or more zones (e.g., detectionregions) for detecting a component in a sample, may be in fluidcommunication with an optional waste area 70 and coupled to outlet 72.In one set of embodiments, fluid may flow in the direction of the arrowsshown in the figure. Further description and examples of such and othercomponents are provided in more detail below.

In some embodiments, sections 71 and 77 of the device are not in fluidcommunication with one another prior to introduction of a sample intothe device. In some cases, sections 71 and 77 are not in fluidcommunication with one another prior to first use of the device, whereinat first use, the sections are brought into fluid communication with oneanother. In other embodiments, however, sections 71 and 77 are in fluidcommunication with one another prior to first use and/or prior tointroduction of a sample into the device. Other configurations ofdevices are also possible.

As shown in the exemplary embodiment illustrated in FIG. 1, one or morefluid flow sources 40 such as a pump and/or a vacuum or otherpressure-control system, valving system 28, detection system 34,temperature regulating system 41, and/or other components may beoperatively associated with one or more of reagent inlet 62, reagentstorage area 64, sample or reagent loading area 66, measurement zone 68,waste area 70, outlet 72, and/or other regions of microfluidic device20. Detection of processes or events in one or more regions of themicrofluidic device can produce a signal or pattern of signals that canbe transmitted to control system 50. Based (at least in part) on thesignal(s) received by the control system, this feedback can be used tomanipulate fluids within and/or between each of these regions of themicrofluidic device, such as by controlling one or more of a pump,vacuum, valving system, detection system, temperature regulating system,and/or other components. In some cases, the feedback can determineabnormalities that have occurred in the microfluidic system, and thecontrol system may send a signal to one or more components to cause allor portions of the system to shut down. Consequently, the quality of theprocesses being performed in the microfluidic system can be controlledusing the systems and methods described herein.

In some embodiments, feedback control involves the detection of one ormore events or processes occurring in a microfluidic system. A varietyof detection methods can be used, as described in more detail below.Detection may involve, for example, determination of at least onecharacteristic of a fluid, a component within a fluid, interactionbetween components within regions of the microfluidic device, or acondition within a region of the microfluidic device (e.g., temperature,pressure, humidity). For instance, detection may involve detecting anopacity of one or more fluids, a concentration of one or more componentsin a fluid, a volume of one or more fluids, a flow rate of one or morefluids, a position of detecting a first fluid in time relative to asecond position in time, and an average time period between thedetection of a first fluid and a second fluid. Detection of the one morecharacteristics, conditions, or events may, in some embodiments, resultin the generation of one or more signals, which can be optionallyfurther processed and transmitted to the control system. As described inmore detail herein, the one or more signals may be compared with one ormore signals, values or thresholds pre-programmed into the controlsystem, and may be used to provide feedback to the microfluidic system.

A variety of signals or patterns of signals can be generated and/ordetermined (e.g., measured) using the systems and methods describedherein. In one set of embodiments, a signal includes an intensitycomponent. Intensity may indicate or be used to indicate, for example,one or more of: the concentration of a component in a fluid, anindication of the type of fluid being detected (e.g., a sample type suchas blood versus urine, or a physical characteristic of the fluid such asa liquid versus a gas), the amount of a component in a fluid, and thevolume of a fluid. In some cases, intensity is determined by an opacityof a fluid or a component. In other embodiments, intensity is determinedby the use of a marker or label such as a fluorescent marker or label.

In some embodiments, a frequency of signals may be generated and/ordetermined For example, a series of signals each having an intensity(e.g., above or below a threshold intensity) may be measured by adetector. This number may be compared with a number of signals or values(having the intensity above or below the threshold intensity)pre-programmed into a control system or other unit. Based at least inpart on this comparison, the control system may initiate, halt, orchange a condition such as the modulation of fluid flow in themicrofluidic system.

In some embodiments, a duration of a signal is generated and/ordetermined The duration of a signal may indicate or be used to indicate,for example, one or more of: the volume of a fluid, the flow rate of afluid, a characteristic of a component within a fluid (e.g., how long acomponent has a certain activity, such as chemiluminescence,fluorescence, and the like), and how long a particular fluid has beenpositioned in a specific region of the microfluidic device.

In some embodiments, a position of a signal in time relative to a secondposition in time or relative to another process or event (e.g., that hasoccurred in the microfluidic system) is generated and/or determined Forexample, a detector may detect when a certain fluid passes across thedetector (e.g., a first position in time), and the timing of this signalmay be related to a second position in time (e.g., when detection wasinitiated; a certain amount of time after a process has occurred, etc.).In another example, a detector may detect when a certain fluid passesacross the detector after (or before) a component of the microfluidicsystem (e.g., a valve) has been actuated. In one embodiment, the openingof a valve may indicate that the mixing of reagents is about to occur,and thus the position of the signal in time may give some indication ofwhen a certain fluid passes across the detector after (or before) themixing of the reagents. If the position of the signal of the fluidoccurs within a certain time range after (or before) the mixing ofreagents, for example, this may indicate that the analysis is runningproperly.

In another example, a detector may detect when a second fluid passesacross the detector after a first fluid has passed across the detector.In other embodiments, a position of a signal in time is determinedrelative to a certain event or process that is taking or has taken placein the microfluidic system (e.g., the start of the analysis, theinitiation of fluid flow, the initiation of detection in themicrofluidic system, upon a user inserting the microfluidic device intoan analyzer, etc.).

In another set of embodiments, an average time between signals or eventsis generated and/or determined. For instance, the average time periodbetween two signals may be measured, where each of the signals mayindependently correspond to one or more characteristics or conditionsdescribed herein. In other embodiments, the average time between thefirst and the last of a series of similar signals is determined (e.g.,the average time between a series of wash fluids passing across adetector).

In certain embodiments, a pattern of signals is generated and/ordetermined. The pattern of signals may include, for example, at leasttwo of (or, in other embodiments, at least three of, or at least fourof) an intensity of a signal, a frequency of signals, a duration of asignal, a position of a signal in time relative to a second position intime or relative to another process or event occurring (or has occurred)in the microfluidic system, and an average time period between two ormore signals or events. In other embodiments, the pattern of signalscomprises at least two of (or, in other embodiments, at least three of,or at least four of) an intensity of a first signal, a duration of thefirst signal, a position of the first signal in time relative to asecond position in time; an intensity of a second signal, a duration ofthe second signal, a position of the second signal in time relative to asecond position in time, and an average time period between the firstand second signals. The pattern of signals may indicate, in someembodiments, whether a particular event or process is taking placeproperly within the microfluidic system. In other embodiments, thepattern of signals indicates whether a particular process or event hasoccurred in the microfluidic system. In yet other embodiments, a patternof signals can indicate a particular sequence of events.

A variety of signals or patterns of signals, such as those describedabove and herein, can be generated and/or determined and can be usedalone or in combination to provide feedback for controlling one more ormore processes, such as modulation of fluid flow in a microfluidicsystem. That is, the control system or any other suitable unit maydetermine, in some embodiments, whether to modulate fluid flow in themicrofluidic system based at least in part on the pattern of signals.For example, determination of whether to modulate fluid flow based atleast in part on a pattern of signals that includes an intensity of afirst signal and a position in time of the first signal relative to asecond position in time may involve the use of both of these pieces ofinformation to make a decision on whether or not to modulate fluid flow.For instance, these signals may be compared to one or more referencesignals (e.g., a threshold intensity or intensity range, and a thresholdposition in time or range of positions in time, relative to a secondposition in time) that may be pre-programmed or pre-set into the controlsystem. If each of the measured signals falls within the respectivethreshold values or ranges, a decision on whether to modulate fluid flowcan be made. Only one of the parameters to be considered (e.g., only anintensity of the first signal or only a position in time of the firstsignal) that meets a threshold value or range may not be sufficientinformation to make a decision on whether or not to modulate fluid flow,because it may not give enough information about the fluid(s) orcomponent(s) that gave rise to the signal(s) for the purposes describedherein. For example, in some cases the fluid or component detected maynot be sufficiently identified for the purposes described herein unlessa pattern of signals is taken into consideration.

In certain embodiments, one or more measured signals is processed ormanipulated (e.g., before or after transmission, and/or before beingcompared to a reference signal or value). It should be appreciated,therefore, that when a signal is transmitted (e.g., to a controlsystem), compared (e.g., with a reference signal or value), or otherwiseused in a feedback process, that the raw signal may be used or aprocessed/manipulated signal based (at least in part) on the raw signalmay be used. For example, in some cases, one or more derivative signalsof a measured signal can be calculated (e.g., using a differentiator, orany other suitable method) and used to provide feedback. In other cases,signals are normalized (e.g., subtracting a measured signal from abackground signal). In one set of embodiments, a signal comprises aslope or average slope, e.g., an average slope of intensity as afunction of time.

In some cases, the measured signal may be converted to a digital signalwith the use of an analog to digital converter so that all furthersignal processing may be performed by a digital computer or digitalsignal processor. Although in one embodiment, all signal processing isperformed digitally, the present invention is not so limited, as analogprocessing techniques may alternatively be used. For instance, a digitalto analog converter may be used to produce an output signal. Signals maybe processed in a time domain (one-dimensional signals), spatial domain(multidimensional signals), frequency domain, autocorrelation domain, orany other suitable domain. In some cases, signals are filtered, e.g.,using a linear filter (a linear transformation of a measured signal), anon-linear filter, a causal filter, a non-causal filter, atime-invariant filter, a time-variant filter, or other suitable filters.It should be understood that the signals, patterns, and their use infeedback described herein are exemplary and that the invention is notlimited in this respect.

Once a signal or pattern of signals has been determined, the signal(s)may be optionally transmitted to a control system. In some cases, thecontrol system compares the signal or pattern of signals to a second setof signal(s). The second signal or pattern of signals may be, forexample, signal(s) determined previously in the microfluidic system, orreference signal(s) or value(s) which may have been pre-programmed intothe control system or other unit of the microfluidic system. In somecases, a reference signal or pattern of signals includes one or morethreshold values or a range of threshold values. The control system maycompare a first signal or pattern of signals with a second signal orpattern of signals (e.g., reference signals), and determine whether toinitiate, cease, or modulate one or more events or series of events inthe microfluidic system. That is, the measured signal or pattern ofsignals can be used by the control system to generate a drive signal andprovide feedback control to the microfluidic system. For example, thecontrol system may determine whether to modulate fluid flow (e.g., flowrate, mixing, the ceasing of flow of one or more fluids) in one or moreregions of the microfluidic system. Other conditions such a modulationof temperature, pressure, humidity, or other conditions can also becontrolled. This modulation may be performed, in certain embodiments, bythe control system sending one or more drive signals to an appropriatecomponent of the microfluidic system (e.g., a valve, pump, vacuum,heater, or other component) to actuate that or another component. Anysuitable valve drive electronics circuit may be used to receive a drivesignal and convert the drive signal to a voltage, current, or othersignal capable of actuating the component. In certain embodiments, thecontrol system can determine whether or not to cease operation of one ormore components of the microfluidic system. In some cases, the controlsystem may determine whether or not to stop an analysis or a portion ofan analysis being conducted in the microfluidic system.

In some embodiments, a method of conducting feedback control may involveinitiating detection of fluids at a first measurement zone of amicrofluidic system. A first fluid and a second fluid may be detected atthe first measurement zone and a first signal corresponding to the firstfluid and a second signal corresponding to the second fluid may beformed. A first pattern of signals may be transmitted to a controlsystem, the first pattern of signals comprising at least two of anintensity of the first signal, a duration of the first signal, aposition of the first signal in time relative to a second position intime, and an average time period between the first and second signals. Adecision about whether to modulate fluid flow in the microfluidic systemmay be determined based at least in part on the first pattern ofsignals.

It should be understood that while much of the description hereindescribes the use of signals or patterns of signals, the invention isnot so limited and that aspects of feedback control or other processesinvolving determination of characteristics, conditions or eventsinvolving fluids or components may not require the generation,determination (e.g., measurement) or analysis of signals or patterns ofsignals in some embodiments.

In some embodiments, a method of conducting feedback involves detectinga first fluid and a second fluid at a first measurement zone of amicrofluidic system, wherein the detection step comprises detecting atleast two of (or at least three of) an opacity of the first fluid, avolume of the first fluid, a flow rate of the first fluid, a position ofthe detection of the first fluid in time relative to a second positionin time, and an average time period between the detection of the firstand second fluids. A decision about whether to modulate fluid flow inthe microfluidic system may be determined based at least in part on thedetection step.

In some embodiments, feedback control can be used to modulate the samecondition, event, or type of condition or event that was first detected.For instance, the concentration of a component in a fluid can bedetermined, and a signal can be generated and transmitted to a controlsystem, which determines whether or not the concentration of the samecomponent should be increased or decreased in the region of themicrofluidic device. In another example, the flow rate of a fluid in achannel is measured, and based at least in part on the signal generatedfrom the measurement, the source of fluid flow (e.g., a vacuum or pump)or a valve is used to modulate the flow rate in that same channel. Insuch and other embodiments, the signal generated may be compared to apre-determined signal or values indicating a desired value or range ofconditions (e.g., concentration, flow rate). The feedback control mayinvolve a feedback loop (e.g., a positive or negative feedback loop) insome cases. In other cases, feedback control does not involve a feedbackloop.

In other embodiments, however, (including many of the examples describedherein) feedback control is based at least in part on the determinationof one or more first conditions or events taking place in themicrofluidic system, and signals from the one or more conditions orevents is used to control a second, different set of conditions orevents taking place (or events that will take place) in the microfluidicsystem. In certain embodiments, the second, different set of conditionsor events do not substantially affect the first set of conditions orevents (e.g., in contrast to the examples of above involving themodulation of concentration of a component or the flow rate in achannel). In some cases, detection takes place at a measurement zone,and feedback from the measurement zone is used to modulate fluid flow ata different region of the microfluidic system. For example, detection ofa certain fluid passing across a detection system may trigger control ofwhether or not a particular valve is actuated to allow flow of one ormore different fluids in a different region of the microfluidic system.In one particular embodiment, detection of a first fluid at (e.g.,passing across) a reaction area may trigger the mixing of second andthird fluids at a mixing region of the microfluidic system. The secondand third fluids may be initially positioned at a different region(e.g., a storage region) of the microfluidic system than from wheredetection and production of the signal used to provide feedback takesplace. In another example, the measurement of optical density of asample flowing across a measurement zone (e.g., a first condition) givesan indication of whether the sample was introduced at the right timeand/or the presence of the correct type or volume of sample. The one ormore signals from this measurement can be compared to one or morepre-set values, and based (at least in part) on this feedback andcomparison, a control system may cease fluid flow in the microfluidicsystem (e.g., a second, different condition) if the measured signalsfalls out of range with the pre-set values. In some such and otherembodiments, the first condition or event has already passed after thedetection step, such that feedback control does not substantiallymodulate that same condition, event, or type of condition or event thatproduced the signal used for feedback.

In some embodiments, one or more feedback control methods such asproportional control, integral control, proportional-integral control,derivative control, proportional-derivative control, integral-derivativecontrol, and proportional-integral-derivative control can be used by acontrol system to modulate fluid flow. The feedback control may involvea feedback loop in some embodiments. In some cases involving one or moreof the aforementioned feedback control methods, a drive signal (whichmay be used to modulate fluid flow, e.g., by actuating a component ofthe microfluidic system) may be generated based at least in part on asignal that is the difference between a pre-programmed threshold signalor value (which may be indicative of a future action to be performed)and a feedback signal that is measured by a detector.

Detection of a condition or an event taking place in a microfluidicsystem may have a variety of forms. In some cases, detection occurscontinuously. In other embodiments, detection occurs periodically; andyet other embodiments, detection occurs sporadically. In some cases,detection occurs upon a specific event or condition taking place.

As described herein, detection can take place at any suitable positionwith respect to a microfluidic device. In some cases, one or moredetectors is stationery with respect to a microfluidic device during useand/or during detection. For example, a stationery detector may bepositioned adjacent a certain region of the microfluidic device, such asa detection region or measurement zone, where one or more events (e.g.,a chemical or biological reaction) takes place. The detector may detect,for example, the passing of fluids across the measurement zone.Additionally or alternatively, the detector may detect the binding orassociation of other components at that region (e.g., the binding of acomponent to surface of the measurement zone). In some embodiments, astationery detector may monitor multiple measurement zonessimultaneously. For example, a detector such as a camera may be used toimage an entire microfluidic device, or large portion of the device, andonly certain areas of the device scrutinized. Components such as opticalfibers may be used to transmit light from multiple measurement zones toa single detector.

In other embodiments, a detector is removably positioned with respect tothe microfluidic device during use and/or during detection. For example,a detector may be physically moved across different regions of themicrofluidic device to detect the movement of fluids across the device.For example, a detector may track the movement of certain fluids and/orcomponents in channels of the microfluidic device. Alternatively, themicrofluidic device can moved relative to a stationary detector. Otherconfigurations and uses of detectors are also possible.

Examples of signals or patterns of signals that can be used in feedbackcontrol are shown in the exemplary embodiment illustrated in FIG. 2.FIG. 2 is a plot showing the detection of various fluids as they flow ina region of a device (e.g., a channel) and pass across a detector. Plot100 shows the measurement of optical density in arbitrary units (y-axis)as a function of time (x-axis). In certain embodiments, the transmissionand/or absorbance of a fluid, for example, can be detected as it passesacross a region of a microfluidic system. An optical density of zero mayindicate maximum light transmission (e.g., low absorbance) and a higheroptical density may indicate low transmission (e.g., higher absorbance).As different fluids flowing across the detector may have differentsusceptibilities to transmission or absorbance of light, the detectionof specific fluids, including their volumes, flow rates, and fluidtypes, can be determined.

For instance, as shown illustratively in FIG. 2, a first fluid producingsignal 110 may pass across the detector at around time=0.1 seconds untilapproximately 700 seconds. (Time=0 seconds may indicate, for example,the initiation of detection.) First fluid 110 has a particular intensity112 (e.g., an optical density of about 0.23). If a particular type offluid having a specific intensity or range of intensities is expected toflow across the detector at a particular point in time (e.g., at a timeof approximately 400 seconds after initiation of detection) or between acertain period of time (e.g., sometime between 0 and 800 seconds), theconfirmation that this process has occurred can be detected. Forexample, first fluid producing 110 may, in some embodiments, be aparticular type of sample that is to be introduced into the microfluidicdevice for performing a particular analysis. If the sample type isassociated with a particular intensity (e.g., whole blood will give anoptical density of approximately 0.23), the type of sample can beverified by determining whether or not that sample has an intensitywithin an allowed range.

Furthermore, the proper introduction of the sample into the device at acorrect time (e.g., at the beginning of the analysis) can be verified bydetermining where the sample signal occurs as a function of time (alongthe x-axis). For instance, the time when the sample reaches themeasurement zone (observed in an OD having a certain range or intensity)can be monitored. If the sample takes too long to enter the measurementzone, this could indicate, for example, a leak or a clog in the system.If it takes too long for the sample to reach the first measurement zoneor there is too much time between the sample or portions of the samplereaching multiple measurement zones (which may be positioned in parallelor in series), the test may be cancelled.

Additionally, the volume of first fluid which produces signal 110 can bedetermined and verified by measuring time period 114 of the signal. Ifthe particular process to be performed in the microfluidic devicerequires a sample having a particular volume, this can be verified. Forexample, a sample having a particular volume (e.g., 10 μL) may beexpected, corresponding to an expected range of flow time (e.g., signalhaving a certain duration) at a certain intensity (e.g., sample OD). Thetest may ensure that the user correctly loaded the sample into thefluidic connector or other suitable sample introduction device. If theduration of the sample signal is too short (which may indicate notenough sample was introduced) or too long (which may indicate too muchsample was introduced) the test may be cancelled and/or the resultsdisregarded.

If, for example, the intensity, time period, or positioning of signal110 that results from the first fluid is incorrect, the control systemmay trigger a secondary process that may, for example, modulate fluidflow in the microfluidic system. For example, in one set of embodiments,the control system may determine that since an incorrect sample type orvolume was introduce into the device, or introduced into the device atan incorrect time, the analysis to be performed by the microfluidicdevice should be canceled. In other embodiments, cancelation may occurdue to a problem with the device (e.g., a clog in the channels that doesnot allow fluid to flow at a particular flow rate), or a problem with ananalyzer used to analyze the device (e.g., the malfunction of one ormore components such as a valve, pump, or vacuum).

The analysis can be canceled, for example, by modulating fluid flow inthe microfluidic system (e.g., sending a signal to a pump or a vacuum tostop the flow of fluids), ceasing power to certain components of thesystem, by ejecting the microfluidic device/cassette from the analyzingsystem (e.g., automatically or informing a user to do so), or by otherprocesses.

In other embodiments, an abnormality occurring in the system triggers asecondary event to occur, but does not cancel the analysis. In somecases, a user may be alerted that an abnormality has occurred in thesystem. The user may be informed that results of the test should not berelied upon, that the analysis needs to be performed again, that theanalysis may take longer to perform, or that the user should take someaction. In some cases, the user can be notified and then asked to verifywhether or not one or more processes of the microfluidic system, or theanalysis being performed, should be continued. Other methods of qualitycontrol are also possible.

In one set of embodiments, a method of conducting quality control todetermine abnormalities in operation of a microfluidic system includesdetecting a first fluid at (e.g., passing across) a first measurementzone of the microfluidic system and forming a first signal correspondingto the first fluid, and transmitting the first signal to a controlsystem. The first signal may be compared to a reference signal, therebydetermining the presence of abnormalities in operation of themicrofluidic system. The method may include determining whether to ceaseoperation of the microfluidic system based at least in part on resultsof the comparing step. In some cases, the control system may determinewhether or not to stop an analysis or a portion of an analysis beingconducted in the microfluidic system.

As show illustratively in FIG. 2, the type of fluid passing across adetector can be determined at least in part by the intensity of thesignal generated by the fluid. For example, whereas signal 110 from afirst fluid has a high intensity (e.g., a low light transmission), asecond series of fluids producing signals 120, 122 and 124 have arelatively low intensity (e.g., a high light transmission). The plotalso indicates the relative separation between the first fluid producingsignal 110 and the second fluids producing signals 120, 122 and 124. Forinstance, the difference between time period 125 and time period 114 cangive an indication of how quickly the second set of fluids is flowedacross the detector after the first fluid has finished passing acrossthe detector. In some embodiments, this difference in time can becompared with one or more reference signals or values (e.g., apredetermined amount of separation time or time range that is supposedto occur between the first fluid and the second fluids). A difference intime that does not match the reference signal or value, or fall withinan allowable range, can indicate that an abnormality has occurred in themicrofluidic system. For example, if the time different between timeperiods 125 and 114 is too long, this may indicate that fluid flow hasbeen obstructed (e.g., due to a clog in a channel by an air bubble or byother means), but later unobstructed in the microfluidic device. In someembodiments, this could influence the test being performed, and as such,the control system may determine whether or not one or more processesshould be ceased or modified in the microfluidic system.

As shown illustratively in FIG. 2, second fluids producing signals 120,122 and 124 are separated by peaks 126, 128 and 130. These peaksrepresent fluids that are flowed between the second fluids. As describedin more detail herein, in some cases these separation fluids may befluids that are immiscible with the fluids they are separating. Forexample, in one set of embodiments, second fluids producing signals 120,122 and 124 are wash solutions that pass across the measurement zone.These wash fluids may be separated by immiscible (separation) fluids(e.g., plugs of air) that produce signals 126, 128 and 130. The washsolutions may have a relatively high transmission, and therefore arelatively low optical density, whereas the plugs of air may have arelatively lower light transmission (e.g., a relatively higher opticaldensity) due to scattering of light as these fluids pass across thedetector. Because of the different susceptibility of these fluids to thetransmission of light, the different fluids (including the fluid type,phase, volume, flow rate) can be distinguished. In addition, thesequence of second fluids passing across the detector may have a timeperiod 134, which may optionally be compared to an optimal time periodor time period range and may optionally be used in feedback control.

In certain embodiments, the number of washes (peaks and troughs) iscounted and a control system cancels the analysis if the expected numberis not observed. Fewer washes could mean the reagents had evaporatedduring storage of the device (indicating a leak) or a problem in theconnection of the fluidic connector. Too few washes could also indicatethat the correct number had not been loaded in the device during devicemanufacture. Too many washes would indicate that the wash plugs hadbroken up during storage.

FIG. 2 also shows a third fluid producing signal 135 passing across themeasurement zone after the flowing of the second fluids. Since the thirdfluid has a similar optical density as those of the second set offluids, the third fluid may be identified or distinguished from otherfluids at least in part by its time period 136, which may give anindication of the fluid's volume. The position of time period 136 alongthe time line (or relative to one or more other signals present) canalso give an indication of the fluid being flowed across the measurementzone. For example, the analysis may be designed such that a fluid givinga certain optical density (e.g., ˜0.01) and duration (e.g., ˜200 secondsat a particular flow rate to be used or pressure to be applied) willoccur between 900 seconds and 1200 seconds after the initiation of theanalysis. These parameters can be pre-programmed into the controlsystem, and compared with signal 135 measured by the detector.

The third fluid producing signal 135 can be any suitable fluid, and insome cases is reagent to be used in a chemical and/or biologicalreaction to be performed in the microfluidic device. For example, asdescribed in more detail below, the third fluid may be a detectionantibody that may bind with one or more components of the sample. Inother embodiments, however, a detection antibody is bound with acomponent of the sample before the sample flows across the detector.Other configurations of binding a detection antibody are also possible,and in some embodiments, no detection antibody is used at all.

After the third fluid is flowed across the measurement zone, a series offourth fluids producing signals 140, 142, 144, 146, 148 and 150 may flowacross the measurement zone. Each of the fourth fluids may be separatedby an immiscible fluid (e.g., air plugs) producing signals 154. Incertain embodiments, the frequency of signals having a certain threshold(e.g., air plugs producing signals 154 having a threshold above anoptical density of 0.05 and/or a series of fourth fluids having anoptical density below 0.01) can be used to trigger one or more events inthe microfluidic system.

In some cases, the intensity and frequency of a series of fluids can becombined with a total time period between the first and last of suchfluids (e.g., time period 158 encompassing the series of fourth fluids).For example, feedback or the triggering of an event may be based atleast in part on the frequency of signals (e.g., peaks) observed incombination with one or more time periods between adjacent signals,and/or in combination with the intensity of the signals, and/or incombination with the time period between the first and last signal ofthat type or intensity. Optionally, one or more of the signals can beused in combination with the average position of the signals relative tothe time scale of events along the time line (e.g., the average time 158between signals 140 and 150 relative to one or more other signals orreference points (e.g., time=zero)).

In some embodiments, the event that is triggered by a pattern of signalsis the modulation of fluid flow within the microfluidic system. Forexample, one or more of a pump, vacuum, valving system, or othercomponent can be actuated based at least in part on the presence ofabsence of a particular pattern of signals. As one example, a pattern ofsignals may trigger the actuation of a valve that allows one or morefluids to flow into a particular channel of the microfluidic device. Forinstance, actuation of the valve may allow two fluids that are keptseparate during storage of the fluids in the device to mix in a commonchannel In one particular embodiment, a mixed fluid includes anamplification reagent that allows amplification of a signal in ameasurement zone of the device. Specific examples are provided in moredetail below.

As described herein, a detector may not only detect the passing offluids across a region of a microfluidic device, but may also detect thepresence or absence of an event or condition occurring in a region ofthe microfluidic device. For example, in some cases a binding event isdetected. In other embodiments, the accumulation and/or deposition of acomponent in a particular region of the microfluidic device is detected.And yet other embodiments, the amplification of a signal is detected.Such processes can occur at any suitable position within a region of adevice. For instance, the event or condition may occur within a fluidpositioned in the region of the device, on a surface of a channel orchamber of the device, on or in a component positioned within the regionof the device (e.g., on a surface of a bead, in a gel, on a membrane).

In some cases, the progression of the event or condition can bedetermined, and, optionally, compared to one or more reference signalsor values (which may be pre-programmed into the control system). Forinstance, as shown illustratively in FIG. 2, a peak 160 may form due tothe build up of a signal (e.g., an opaque layer) in a measurement zone.This slope of the peak may be measured and compared with one or morecontrol values to determine whether or not a correct process isoccurring or has occurred in the measurement zone. For example, if theslope of peak 160 is within a particular range of acceptable values,this may indicate that there were no abnormalities in the storage ofreagents that were used in part to produce the signal.

In one set of embodiments, peak 160 indicates an amplification reagententering the measurement zone. The analysis may be designed andconfigured such that the amplification reagent enters the measurementzone within a certain time period after a certain event takes place(e.g., upon actuation of a valve). In some cases, the amplificationreagent should have a certain optical density associated with it (e.g.,a low optical density if the reagent is a clear liquid). If the reagentis late in arriving at the measurement zone and/or or the initialoptical density is too high, the test can be cancelled. If the reagenthas a high optical density (e.g., it is dark or opaque), this couldindicate that the reagent has been spoiled (e.g., during storage of thereagent in the device).

In some embodiments, a device may include multiple measurement zones(e.g., in parallel or in series). One measurement zone may be used as anegative control. For instance, minimal binding or deposition of asubstance (e.g., an opaque layer), and therefore a low optical densityin some embodiments, may be expected in the negative control measurementzone. If a detector measures an elevated optical density in the negativecontrol measurement zone, this may indicate, for example, non-specificbinding. In some cases, the signal from this measurement zone can beconsidered “background” and subtracted from signals in the othermeasurement zones to account for non-specific binding which may occurthroughout the system. If the background is too high, the test may becancelled. This may, for example, indicate a problem with theamplification reagents or other reagents used in the analysis.

In some embodiments, a device may include a measurement zone used as apositive control. The positive control may, in some embodiments, includea known amount of analyte bound to the measurement zone (e.g., to thechannel walls), and the level of the optical density signals at acertain point in time, the slope of these signals, or the change inslope of these signals in the zone may fall within an expected range.These ranges can be determined during calibration of a specified lot ofdevices. In some cases, as described in more detail herein, thisinformation may be included in the lot-specific information transferredto an analyzer by use of a lot-specific tag, such as a bar-code, memorystick, or radio-frequency identification (RFID) tag. If the referencelevels for these measurement zones fall out of range, the test may becancelled. Similar to background, these signals can also be used toadjust the test signal (e.g., increasing the test signal slightly ifthese signals are elevated, decreasing the test signal if these signalsare low).

The presence of obstructions such as bubbles or other components duringone or more events (e.g., amplification, mixing) and/or at one or moreunexpected positions in time may indicate problems in the analysis, suchas a leak in a valve. These bubbles or other components can be detectedas peaks having a certain intensity in the optical density pattern(which may be similar to the air plug peaks used during washing). Ifthese are observed in unexpected places, the test can be cancelled.

It should be understood that while optical density (e.g., transmissionor absorbance) was determined in FIG. 2, in other embodiments othertypes of signals can be measured using a suitable detector. The signalsmay be produced absent a label (such as in measuring optical density),or produced using a label. A variety of different labels can be used,such as fluorescent markers, dyes, quantum dots, magnetic particles, andother labels known in the art.

As shown illustratively in FIG. 2, in some embodiments an analysisperformed in a device can be recorded to produce essentially a“fingerprint” of the analysis. All or potions of the fingerprint may beused to provide feedback to the microfluidic system. In some cases, thefingerprint includes signals from the passing of substantially allfluids used in an analysis across a region of the device. Sincedifferent fluids used in the analysis may have different volumes, flowrates, compositions, and other characteristics, these properties can bereflected in the fingerprint. As such, the fingerprint can be used toidentify, for example, the fluids used in the analysis, the timing ofthe fluids (e.g., when particular fluids were introduced into certainregions of the device), interaction of the fluids (e.g., mixing). Insome embodiments, the fingerprint can be used to identify the type ofanalysis performed in the device and/or the test format (e.g., asandwich assay versus a competitive assay) of the analysis.

In one set of embodiments, the fingerprint as a whole (e.g., the generalshape, duration, and timing of all signals) is used to conduct qualitycontrol at the end of the analysis. For instance, the fingerprint may becompared with a control fingerprint to determine whether the analysiswas run properly after all fluids have been flowed. The control systemmay, in some cases, notify the user as to whether the analysis was runproperly (e.g., via a user interface).

In other embodiments, a detector may be positioned within certainregions of a microfluidic system and may only determine the presence orpassing of certain, but not all, fluids across the detector. Forexample, a detector may be positioned at a mixing region to determineproper mixing of fluids. If the fluids are mixed properly (e.g., a mixedfluid having a certain property such as a certain concentration orvolume is produced) or mixed at a proper point in time relative to oneor more other events occurring in the analysis, feedback control mayallow the mixed fluid to flow into another region of the device. If themixed fluid does not have one or more desired or predeterminedcharacteristics, feedback control may prevent the mixed fluid mayflowing into the region and, in some embodiments, may initiate a secondset of fluids to be mixed and transported to the region.

In certain embodiments, feedback control comprises the use of two ormore detectors. A first detector may determine a first set of signals,and a second detector may determine a second set of signals. The firstand second set of signals may be compared with one another, and/or eachmay be compared with a set of reference signals or values which may bepre-programmed into a control system. For example, a device may includea plurality of measurement zones, each measurement zone associated witha detector that measures signals in that region. In some cases, thesystem is designed and configured such that a first detector determinesa fingerprint of the analysis that substantially matches the fingerprintof the analysis of a second detector. If the fingerprints do not match,however, this may indicate that an abnormality has occurred within thesystem. In some cases, the first and/or second detectors may detect thepassing of all fluids used in the analysis across a region of thedevice, or only certain (but not all) fluids passing across a region ofthe device, as described above. In other embodiments, feedback control,or determination of a value in general, may involve the use of signalsdetected from multiple measurement zones. For example, flow rate may bedetermined by measuring how long it takes a bubble or a leading edge ofa fluid to travel between two measurement zones.

Feedback control and other processes and methods described herein may beconducted using any suitable microfluidic system, such as thosedescribed in more detail below. In some cases, the microfluidic systemincludes a device or cassette that may be configured to be inserted in amicrofluidic sample analyzer. FIGS. 3-6 illustrate various exemplaryembodiments of the cassette 20 for use with an analyzer. As shownillustratively in these figures, the cassette 20 may be substantiallycard-shaped (i.e. similar to a card key) having a substantially rigidplate-like structure. Non-limiting examples of microfluidic systems thatcan be part of cassette and that can be used with the systems andmethods described herein are described in more detail in InternationalPatent Publication No. WO2005/066613 (International Patent ApplicationSerial No. PCT/US2004/043585), filed Dec. 20, 2004 and entitled “AssayDevice and Method” and U.S. patent application Ser. No. 12/113,503,published as U.S. Patent Publication No. 2008/0273918, filed May 1, 2008and entitled “Fluidic Connectors and Microfluidic Systems”, each ofwhich are incorporated herein by reference in their entireties for allpurposes.

The cassette 20 may be configured to include a fluidic connector 220,which as shown in exemplary embodiment illustrated in FIG. 3, may snapinto one end of the cassette 20. In certain embodiments, the fluidicconnector can be used to introduce one or more fluids (e.g., a sample ora reagent) into the cassette.

In one set of embodiments, the fluidic connector is used to fluidlyconnect two (or more) channels of the cassette during first use, whichchannels are not connected prior to first use. For example, the cassettemay include two channels that are not in fluid communication prior tofirst use of the cassette. Non-connected channels may be advantageous incertain cases, such as for storing different reagents in each of thechannels. For example, a first channel may be used to store dry reagentsand a second channel may be used to store wet reagents. Having thechannels be physically separated from one another can enhance long-termstability of the reagents stored in each of the channels, e.g., bykeeping the reagent(s) stored in dry form protected from moisture thatmay be produced by reagent(s) stored in wet form. At first use, thechannels may be connected via the fluidic connector to allow fluidcommunication between the channels of the cassette. For instance, thefluidic connected may puncture seals covering inlets and/or outlets ofthe cassette to allow insertion of the fluidic connector into thecassette.

As used herein, “prior to first use of the cassette” means a time ortimes before the cassette is first used by an intended user aftercommercial sale. First use may include any step(s) requiringmanipulation of the device by a user. For example, first use may involveone or more steps such as puncturing a sealed inlet to introduce areagent into the cassette, connecting two or more channels to causefluid communication between the channels, preparation of the device(e.g., loading of reagents into the device) before analysis of a sample,loading of a sample onto the device, preparation of a sample in a regionof the device, performing a reaction with a sample, detection of asample, etc. First use, in this context, does not include manufacture orother preparatory or quality control steps taken by the manufacturer ofthe cassette. Those of ordinary skill in the art are well aware of themeaning of first use in this context, and will be able easily todetermine whether a cassette of the invention has or has not experiencedfirst use. In one set of embodiments, cassette of the invention aredisposable after first use (e.g., after completion of an assay), and itis particularly evident when such devices are first used, because it istypically impractical to use the devices at all (e.g., for performing asecond assay) after first use.

A cassette may be coupled to a fluidic connector using a variety ofmechanisms. For example, the fluidic connector may include at least onenon-fluidic feature complementary to a feature of the cassette so as toform a non-fluidic connection between the fluidic connector and thecassette upon attachment. The non-fluidic complementary feature may be,for example, a protruding feature of the fluidic connector andcorresponding complementary cavities of the cassette, which can help theuser align the fluidic connector with the cassette. In some cases, thefeature creates a substantial resistance to movement of the fluidicconnector relative to the cassette and/or alignment element upon thealignment element receiving the fluidic component (e.g., upon insertionof the fluidic component into the alignment element) and/or duringintended use of the device. The fluidic connector and/or cassette mayoptionally include one or more features such as snap features (e.g.,indentations), grooves, openings for inserting clips, zip-tiemechanisms, pressure-fittings, friction-fittings, threaded connectorssuch as screw fittings, snap fittings, adhesive fittings, magneticconnectors, or other suitable coupling mechanisms. These and otherexamples of coupling mechanisms are described in more detail in U.S.patent application Ser. No. 12/113,503, published as U.S. PatentPublication No. 2008/0273918, filed May 1, 2008 and entitled “FluidicConnectors and Microfluidic Systems”, which is incorporated herein byreference in its entirety for all purposes. Connection of the fluidicconnector to the cassette may involve forming a liquid-tight and/orair-tight seal between the components. Attachment of a fluidic connectorto a cassette may be reversible or irreversible.

As shown, the cassette 20 may be configured to include a fluidicconnector 220. In particular, the cassette 20 may include a fluidicconnector alignment element 202 which is configured to receive and matewith the connector 220. The alignment element may be constructed andarranged to engage with the fluidic connector and thereby position theconnector in a predetermined, set configuration relative to thecassette. As shown in the illustrative embodiments of FIG. 3, thecassette may include an alignment element that extends approximatelyperpendicular to the cassette. In other embodiments, the alignmentelement may extend approximately parallel to the cassette. Examples ofalignment elements are described in more detail in U.S. patentapplication Ser. No. 12/113,503, published as U.S. Patent PublicationNo. 2008/0273918, filed May 1, 2008 and entitled “Fluidic Connectors andMicrofluidic Systems”, which is incorporated herein by reference in itsentirety for all purposes.

In some embodiments, the configuration of the alignment element and thefluidic connector may be adapted to allow insertion of the fluidicconnector into the alignment element by a sliding motion. For example,the fluidic connector may slide against one or more surfaces of thealignment element when the fluidic connector is inserted into thealignment element.

The fluidic connector may include a substantially U-shaped channel whichmay hold a fluid and/or reagent (e.g., a fluid sample) prior to beconnected to the cassette. The channel may be housed between two shellcomponents which form the connector. In some embodiments, the fluidicconnector may be used to collect a sample from the patient prior to thefluidic connector being connected to the cassette. For example, with ablood sample, the fluidic connector may be configured to puncture apatient's finger to collect the sample in the channel In otherembodiments, fluid connector does not contain a sample (or reagent)prior to connection to the cassette, but simply allows fluidcommunication between two or more channels of the cassette uponconnection. In one embodiment, the U-shaped channel is formed with acapillary tube. The fluidic connector can also include other channelconfigurations, and in some embodiments, may include more than onechannels that may be fluidically connected or unconnected to oneanother.

As shown illustratively in the exploded assembly view of FIG. 4, thecassette 20 may include a cassette body 204 which includes at least onechannel 206 configured to receive a sample or reagent. The cassette body204 may also include latches 208 positioned on one end that interlockwith the fluidic connector alignment element 202 for a snap fit.

The cassette 20 may also include top and bottom covers 210 and 212,which may, for example, be made of a transparent material. In someembodiments, a cover can be in the form of a biocompatible adhesive andcan be made of a polymer (e.g., PE, COC, PVC) or an inorganic materialfor example. In some cases, one or more covers are in the form of anadhesive film (e.g., a tape). For some applications, the material anddimensions of a cover are chosen such that the cover is substantiallyimpermeable to water vapor. In other embodiments, the cover can benon-adhesive, but may bond thermally to the microfluidic substrate bydirect application of heat, laser energy, or ultrasonic energy. Anyinlet(s) and/or outlet(s) of a channel of the cassette can be sealed(e.g., by placing an adhesive over the inlet(s) and/or outlet(s)) usingone or more covers. In some cases, the cover substantially seals one ormore stored reagents in the cassette.

As illustrated, the cassette body 204 may include one or more ports 214coupled to the channel 206 in the cassette body 204. These ports 214 canbe configured to align with the substantially U-shaped channel 222 inthe fluidic connector 220 when the fluidic connector 220 is coupled tothe cassette 20 to fluidly connect the channel 206 in the cassette body204 with the channel 222 in the fluidic connector 220. As shown, a cover216 may be provided over the ports 214 and the cover 216 may beconfigured to be pieced or otherwise opened (e.g., by the connector 220or by other means) to fluidly connect the two channels 206 and 222.Additionally, a cover 218 may be provided to cover port 219 (e.g., avacuum port) in the cassette body 204. As set forth in further detailbelow, the port 219 may be configured to fluidly connect a fluid flowsource 40 with the channel 206 to move a sample through the cassette.The cover 218 over the port 219 may be configured to be pierced orotherwise opened to fluidly connect the channel 206 with the fluid flowsource 40.

The cassette body 204 may optionally include a liquid containment regionsuch as a waste area, including an absorbent material 217 (e.g., a wastepad). In some embodiments, the liquid containment region includesregions that capture one or more liquids flowing in the cassette, whileallowing gases or other fluids in the cassette to pass through theregion. This may be achieved, in some embodiments, by positioning one ormore absorbent materials in the liquid containment region for absorbingthe liquids. This configuration may be useful for removing air bubblesfrom a stream of fluid and/or for separating hydrophobic liquids fromhydrophilic liquids. In certain embodiments, the liquid containmentregion prevents liquids from passing through the region. In some suchcases, the liquid containment region may act as a waste area bycapturing substantially all of the liquid in the cassette, therebypreventing liquids from exiting the cassette (e.g., while allowing gasesto escape from an outlet of the cassette). For example, the waste areamay be used to store the sample and/or reagents in the cassette afterthey have passed through the channel 206 during the analysis of thesample. These and other arrangements may be useful when the cassette isused as a diagnostic tool, as the liquid containment region may preventa user from being exposed to potentially-harmful fluids in the cassette.Non-limiting examples of liquid containment regions are described inmore detail in U.S. patent applcation Ser. No. 12/196,392, published asU.S. Patent Publication No. 2009/0075390, filed Aug. 22, 2008, entitled“Liquid containment for integrated assays”, which is incorporated hereinby reference in its entirety for all purposes.

FIG. 5 shows a cassette having a certain configuration of channels andincluding various components of a microfluidic system for manipulatingfluids. FIG. 6 shows another example of a configuration of channels thatmay be part of a cassette. As shown illustratively in FIGS. 5 and 6, insome embodiments, a cassette may include a first channel 206 and asecond channel 207 spaced apart from the first channel In oneembodiment, the channels 206, 207 range in largest cross-sectiondimension from approximately 50 micrometers to approximately 500micrometers, although other channel sizes and configurations may beused, as described in more detail below.

The first channel 206 may include one or more measurement zones used toanalyze the sample. For example, in one illustrative embodiment, thechannel 206 includes four measurement zones 209 which are utilizedduring sample analysis (see FIG. 6).

In certain embodiments, one or more measurement zones are the form ofmeandering regions (e.g., involving meandering channels), as describedin more detail below and in International Patent Publication No.WO2006/113727 (International Patent Application Serial No.PCT/US06/14583), filed Apr. 19, 2006 and entitled “Fluidic StructuresIncluding Meandering and Wide Channels”; U.S. patent application Ser.No. 12/113,503, published as U.S. Patent Publication No. 2008/0273918,filed May 1, 2008 and entitled “Fluidic Connectors and MicrofluidicSystems” and U.S. patent application Ser. No. 12/196,392, published asU.S. Patent Publication No. 2009/0075390, filed Aug. 22, 2008, entitled“Liquid containment for integrated assays”, each of which isincorporated herein by reference in their entireties for all purposes. Ameandering region may, for example, be defined by an area of at least0.25 mm², at least 0.5 mm², at least 0.75 mm², or at least 1.0 mm²,wherein at least 25%, 50%, or 75% of the area of the meandering regioncomprises an optical detection pathway. A detector that allowsmeasurement of a single signal through more than one adjacent segmentsof the meandering region may be positioned adjacent the meanderingregion.

As described herein, the first channel 206 and/or the second channel 207may be used to store one or more reagents used to process and analyzethe sample prior to first use of the cassette. In some embodiments, dryreagents are stored in one channel or section of a cassette and wetreagents are stored in a second channel or section of cassette.Alternatively, two separate sections or channels of a cassette may bothcontain dry reagents and/or wet reagents. Reagents can be stored and/ordisposed, for example, as a liquid, a gas, a gel, a plurality ofparticles, or a film. The reagents may be positioned in any suitableportion of a cassette, including, but not limited to, in a channel,reservoir, on a surface, and in or on a membrane, which may optionallybe part of a reagent storage area. A reagent may be associated with acassette (or components of a cassette) in any suitable manner. Forexample, reagents may be crosslinked (e.g., covalently or ionically),absorbed, or adsorbed (physisorbed) onto a surface within the cassette.In one particular embodiment, all or a portion of a channel (such as afluid path of a fluid connector or a channel of the cassette) is coatedwith an anti-coagulant (e.g., heparin). In some cases, a liquid iscontained within a channel or reservoir of a cassette prior to first useand/or prior to introduction of a sample into the cassette.

In some embodiments, the stored reagents may include fluid plugspositioned in linear order so that during use, as fluids flow to areaction site, they are delivered in a predetermined sequence. Acassette designed to perform an assay, for example, may include, inseries, a rinse fluid, a labeled-antibody fluid, a rinse fluid, and aamplification fluid, all stored therein. While the fluids are stored,they may be kept separated by substantially immiscible separation fluids(e.g., a gas such as air) so that fluid reagents that would normallyreact with each other when in contact may be stored in a common channel.

Reagents can be stored in a cassette for various amounts of time. Forexample, a reagent may be stored for longer than 1 hour, longer than 6hours, longer than 12 hours, longer than 1 day, longer than 1 week,longer than 1 month, longer than 3 months, longer than 6 months, longerthan 1 year, or longer than 2 years. Optionally, the cassette may betreated in a suitable manner in order to prolong storage. For instance,cassettes having stored reagents contained therein may be vacuum sealed,stored in a dark environment, and/or stored at low temperatures (e.g.,below 0 degrees C.). The length of storage depends on one or morefactors such as the particular reagents used, the form of the storedreagents (e.g., wet or dry), the dimensions and materials used to formthe substrate and cover layer(s), the method of adhering the substrateand cover layer(s), and how the cassette is treated or stored as awhole.

As illustrated in the exemplary embodiment shown in FIGS. 5 and 6,channels 206 and 207 may not be in fluid communication with each otheruntil the fluidic connector 220 is coupled to the cassette 20. In otherwords, the two channels, in some embodiments, are not in fluidcommunication with one another prior to first use and/or prior tointroduction of a sample into the cassette. In particular, asillustrated, the substantially U-shaped channel 222 of the connector 220may fluidly connect the first and second channels 206, 207 such that thereagents in the second channel 207 can pass through the U-shaped channel22 and selectively move into the measurement zones 209 in the firstchannel 206. In other embodiments, the two channels 206 and 207 are influid communication with one another prior to first use, and/or prior tointroduction of a sample into the cassette, but the fluidic connectorfurther connects the two channels (e.g., to form a closed-loop system)upon first use.

In some embodiments, a cassette described herein may include one moremicrofluidic channels, although such cassettes are not limited tomicrofluidic systems and may relate to other types of fluidic systems.“Microfluidic,” as used herein, refers to a cassette, device, apparatusor system including at least one fluid channel having a maximumcross-sectional dimension of less than 1 mm, and a ratio of length tolargest cross-sectional dimension of at least 3:1. A “microfluidicchannel,” as used herein, is a channel meeting these criteria.

The “cross-sectional dimension” (e.g., a diameter) of the channel ismeasured perpendicular to the direction of fluid flow. Most fluidchannels in components of cassettes described herein have maximumcross-sectional dimensions less than 2 mm, and in some cases, less than1 mm In one set of embodiments, all fluid channels of a cassette aremicrofluidic or have a largest cross sectional dimension of no more than2 mm or 1 mm In another set of embodiments, the maximum cross-sectionaldimension of the channel(s) are less than 500 microns, less than 200microns, less than 100 microns, less than 50 microns, or less than 25microns. In some cases the dimensions of the channel may be chosen suchthat fluid is able to freely flow through the article or substrate. Thedimensions of the channel may also be chosen, for example, to allow acertain volumetric or linear flowrate of fluid in the channel. Ofcourse, the number of channels and the shape of the channels can bevaried by any suitable method known to those of ordinary skill in theart. In some cases, more than one channel or capillary may be used.

A channel may include a feature on or in an article (e.g., a cassette)that at least partially directs the flow of a fluid. The channel canhave any suitable cross-sectional shape (circular, oval, triangular,irregular, square or rectangular, or the like) and can be covered oruncovered. In embodiments where it is completely covered, at least oneportion of the channel can have a cross-section that is completelyenclosed, or the entire channel may be completely enclosed along itsentire length with the exception of its inlet(s) and outlet(s). Achannel may also have an aspect ratio (length to average cross sectionaldimension) of at least 2:1, more typically at least 3:1, 5:1, or 10:1 ormore.

Cassettes described herein may include channels or channel segmentspositioned on one or two sides of the cassette. In some cases, thechannels are formed in a surface of the cassette. The channel segmentsmay be connected by an intervening channel passing through the cassette.Non-limiting examples of such and other channel configurations aredescribed in more detail in U.S. patent application Ser. No. 12/640,420,filed Dec. 17, 2009, entitled, “Reagent Storage in Microfluidic Systemsand Related Articles and Methods”, which is incorporated herein byreference in its entirety for all purposes. In some embodiments, thechannel segments are used to store reagents in the device prior to firstuse by an end user. The specific geometry of the channel segments andthe positions of the channel segments within the cassettes may allowfluid reagents to be stored for extended periods of time without mixing,even during routine handling of the cassettes such as during shipping ofthe cassettes, and when the cassettes are subjected to physical shock orvibration.

In certain embodiments, a cassette includes optical elements that arefabricated on one side of a cassette opposite a series of fluidicchannels. An “optical element” is used to refer to a feature formed orpositioned on or in an article or cassette that is provided for and usedto change the direction (e.g., via refraction or reflection), focus,polarization, and/or other property of incident electromagneticradiation relative to the light incident upon the article or cassette inthe absence of the element. For example, an optical element may comprisea lens (e.g., concave or convex), mirror, grating, groove, or otherfeature formed or positioned in or on a cassette. A cassette itselfabsent a unique feature, however, would not constitute an opticalelement, even though one or more properties of incident light may changeupon interaction with the cassette. The optical elements may guideincident light passing through the cassette such that most of the lightis dispersed away from specific areas of the cassette, such asintervening portions between the fluidic channels. By decreasing theamount of light incident upon these intervening portions, the amount ofnoise in a detection signal can be decreased when using certain opticaldetection systems. In some embodiments, the optical elements comprisetriangular grooves formed on or in a surface of the cassette. The draftangle of the triangular grooves may be chosen such that incident lightnormal to the surface of the cassette is redirected at an angledependent upon the indices of refraction of the external medium (e.g.,air) and the cassette material. In some embodiments, one or more opticalelements are positioned between adjacent segments of a meandering regionof a measurement zone. Non-limiting examples of optical elements andconfigurations of channels and components with respect to the opticalelements are described in more detail in U.S. patent application Ser.No. 12/698,451, filed Feb. 2, 2010, entitled, “Structures forControlling Light Interaction with Microfluidic Devices”, which isincorporated herein by reference in its entirety for all purposes.

A cassette can be fabricated of any material suitable for forming achannel Non-limiting examples of materials include polymers (e.g.,polyethylene, polystyrene, polymethylmethacrylate, polycarbonate,poly(dimethylsiloxane), PTFE, PET, and a cyclo-olefin copolymer), glass,quartz, and silicon. The material forming the cassette and anyassociated components (e.g., a cover) may be hard or flexible. Those ofordinary skill in the art can readily select suitable material(s) basedupon e.g., its rigidity, its inertness to (e.g., freedom fromdegradation by) a fluid to be passed through it, its robustness at atemperature at which a particular device is to be used, itstransparency/opacity to light (e.g., in the ultraviolet and visibleregions), and/or the method used to fabricate features in the material.For instance, for injection molded or other extruded articles, thematerial used may include a thermoplastic (e.g., polypropylene,polycarbonate, acrylonitrile-butadiene-styrene, nylon 6), an elastomer(e.g., polyisoprene, isobutene-isoprene, nitrile, neoprene,ethylene-propylene, hypalon, silicone), a thermoset (e.g., epoxy,unsaturated polyesters, phenolics), or combinations thereof.

In some embodiments, the material and dimensions (e.g., thickness) of acassette and/or cover are chosen such that it is substantiallyimpermeable to water vapor. For instance, a cassette designed to storeone or more fluids therein prior to first use may include a covercomprising a material known to provide a high vapor barrier, such asmetal foil, certain polymers, certain ceramics and combinations thereof.In other cases, the material is chosen based at least in part on theshape and/or configuration of the cassette. For instance, certainmaterials can be used to form planar devices whereas other materials aremore suitable for forming devices that are curved or irregularly shaped.

In some instances, a cassette is comprised of a combination of two ormore materials, such as the ones listed above. For instance, channels ofthe cassette may be formed in polystyrene or other polymers (e.g., byinjection molding) and a biocompatible tape may be used to seal thechannels. The biocompatible tape or flexible material may include amaterial known to improve vapor barrier properties (e.g., metal foil,polymers or other materials known to have high vapor barriers), and mayoptionally allow access to inlets and outlets by puncturing or unpeelingthe tape. A variety of methods can be used to seal a microfluidicchannel or portions of a channel, or to join multiple layers of adevice, including but not limited to, the use of adhesives, use adhesivetapes, gluing, bonding, lamination of materials, or by mechanicalmethods (e.g., clamping, snapping mechanisms, etc.).

In some instances, a cassette comprises a combination of two or moreseparate layers (or cassettes) mounted together. Independent channelnetworks (such as sections 71 and 77 of FIG. 1A), which may optionallyinclude reagents stored therein prior to first use, may be included onseparate layers (or cassettes). The separate layers may be mountedtogether by any suitable means, such as by the methods described herein,to form a single cassette. In some embodiments, two or more channelnetworks are connected fluidically at first use, e.g., by use of afluidic connector. In other embodiments, two or more channel networksare connected fluidically prior to first use.

A cassette described herein may have any suitable volume for carryingout an analysis such as a chemical and/or biological reaction or otherprocess. The entire volume of a cassette includes, for example, anyreagent storage areas, measurement zones, liquid containment regions,waste areas, as well as any fluid connectors, and fluidic channelsassociated therewith. In some embodiments, small amounts of reagents andsamples are used and the entire volume of the fluidic device is, forexample, less than 10 mL, 5 mL, 1 mL, 500 μL, 250 μL, 100 μL, 50 μL, 25μL, 10 μL, 5 ρL, or 1 μL.

A cassette described herein may be portable and, in some embodiments,handheld. The length and/or width of the cassette may be, for example,less than or equal to 20 cm, 15 cm, 10 cm, 8 cm, 6 cm, or 5 cm. Thethickness of the cassette may be, for example, less than or equal to 5cm, 3 cm, 2 cm, 1 cm, 8 mm, 5 mm, 3 mm, 2 mm, or 1 mm Advantageously,portable devices may be suitable for use in point-of-care settings.

It should be understood that the cassettes and their respectivecomponents described herein are exemplary and that other configurationsand/or types of cassettes and components can be used with the systemsand methods described herein.

The methods and systems described herein may involve variety ofdifferent types of analyses, and can be used to determine a variety ofdifferent samples. In some cases, an analysis involves a chemical and/orbiological reaction. In some embodiments, a chemical and/or biologicalreaction involves binding. Different types of binding may take place incassettes described herein. Binding may involve the interaction betweena corresponding pair of molecules that exhibit mutual affinity orbinding capacity, typically specific or non-specific binding orinteraction, including biochemical, physiological, and/or pharmaceuticalinteractions. Biological binding defines a type of interaction thatoccurs between pairs of molecules including proteins, nucleic acids,glycoproteins, carbohydrates, hormones and the like. Specific examplesinclude antibody/antigen, antibody/hapten, enzyme/substrate,enzyme/inhibitor, enzyme/cofactor, binding protein/substrate, carrierprotein/substrate, lectin/carbohydrate, receptor/hormone,receptor/effector, complementary strands of nucleic acid,protein/nucleic acid repressor/inducer, ligand/cell surface receptor,virus/ligand, etc. Binding may also occur between proteins or othercomponents and cells. In addition, devices described herein may be usedfor other fluid analyses (which may or may not involve binding and/orreactions) such as detection of components, concentration, etc.

In some cases, a heterogeneous reaction (or assay) may take place in acassette; for example, a binding partner may be associated with asurface of a channel, and the complementary binding partner may bepresent in the fluid phase. Other solid-phase assays that involveaffinity reaction between proteins or other biomolecules (e.g., DNA,RNA, carbohydrates), or non-naturally occurring molecules, can also beperformed. Non-limiting examples of typical reactions that can beperformed in a cassette include chemical reactions, enzymatic reactions,immuno-based reactions (e.g., antigen-antibody), and cell-basedreactions.

Non-limiting examples of analytes that can be determined (e.g.,detected) using cassettes described herein include specific proteins,viruses, hormones, drugs, nucleic acids and polysaccharides;specifically antibodies, e.g., IgD, IgG, IgM or IgA immunoglobulins toHTLV-I, HIV, Hepatitis A, B and non A/non B, Rubella, Measles, HumanParvovirus B19, Mumps, Malaria, Chicken Pox or Leukemia; human andanimal hormones, e.g., thyroid stimulating hormone (TSH), thyroxine(T4), luteinizing hormone (LH), follicle-stimulating hormones (FSH),testosterone, progesterone, human chorionic gonadotropin, estradiol;other proteins or peptides, e.g. troponin I, c-reactive protein,myoglobin, brain natriuretic protein, prostate specific antigen (PSA),free-PSA, complexed-PSA, pro-PSA, EPCA-2, PCADM-1, ABCAS, hK2, beta-MSP(PSP94), AZGP1, Annexin A3, PSCA, PSMA, JM27, PAP; drugs, e.g.,paracetamol or theophylline; marker nucleic acids, e.g., PCA3,TMPRS-ERG; polysaccharides such as cell surface antigens for HLA tissuetyping and bacterial cell wall material. Chemicals that may be detectedinclude explosives such as TNT, nerve agents, and environmentallyhazardous compounds such as polychlorinated biphenyls (PCBs), dioxins,hydrocarbons and MTBE. Typical sample fluids include physiologicalfluids such as human or animal whole blood, blood serum, blood plasma,semen, tears, urine, sweat, saliva, cerebro-spinal fluid, vaginalsecretions; in-vitro fluids used in research or environmental fluidssuch as aqueous liquids suspected of being contaminated by the analyte.

In some embodiments, one or more reagents that can be used to determinean analyte of a sample (e.g., a binding partner of the analyte to bedetermined) is stored in a channel or chamber of a cassette prior tofirst use in order to perform a specific test or assay. In cases wherean antigen is being analyzed, a corresponding antibody or aptamer can bethe binding partner associated with a surface of a microfluidic channelIf an antibody is the analyte, then an appropriate antigen or aptamermay be the binding partner associated with the surface. When a diseasecondition is being determined, it may be preferred to put the antigen onthe surface and to test for an antibody that has been produced in thesubject. Such antibodies may include, for example, antibodies to HIV.

In some embodiments, a cassette is adapted and arranged to perform ananalysis involving accumulating an opaque material on a region of amicrofluidic channel, exposing the region to light, and determining thetransmission of light through the opaque material. An opaque materialmay include a substance that interferes with the transmittance of lightat one or more wavelengths. An opaque material does not merely refractlight, but reduces the amount of transmission through the material by,for example, absorbing or reflecting light. Different opaque materialsor different amounts of an opaque material may allow transmittance ofless than, for example, 90, 80, 70, 60, 50, 40, 30, 20, 10 or 1 percentof the light illuminating the opaque material. Examples of opaquematerials include molecular layers of metal (e.g., elemental metal),ceramic layers, polymeric layers, and layers of an opaque substance(e.g., a dye). The opaque material may, in some cases, be a metal thatcan be electrolessly deposited. These metals may include, for example,silver, copper, nickel, cobalt, palladium, and platinum.

An opaque material that forms in a channel may include a series ofdiscontinuous independent particles that together form an opaque layer,but in one embodiment, is a continuous material that takes on agenerally planar shape. The opaque material may have a dimension (e.g.,a width of length) of, for example, greater than or equal to 1 micron,greater than or equal to 5 microns, greater than 10 microns, greaterthan or equal to 25 microns, or greater than or equal to 50 microns. Insome cases, the opaque material extends across the width of the channel(e.g., a measurement zone) containing the opaque material. The opaquelayer may have a thickness of, for example, less than or equal to 10microns, less than or equal to 5 microns, less than or equal to 1micron, less than or equal to 100 nanometers or less than or equal to 10nanometers. Even at these small thicknesses, a detectable change intransmittance can be obtained. The opaque layer may provide an increasein assay sensitivity when compared to techniques that do not form anopaque layer.

In one set of embodiments, a cassette described herein is used forperforming an immunoassay (e.g., for human IgG or PSA) and, optionally,uses silver enhancement for signal amplification. A cassette describedherein may have one or more similar characteristics as those describedin U.S. patent application Ser. No. 12/113,503, published as U.S. PatentPublication No. 2008/0273918, filed May 1, 2008 and entitled “FluidicConnectors and Microfluidic Systems”, which is incorporated herein byreference. In such an immunoassay, after delivery of a sample containinghuman IgG to a reaction site or analysis region, binding between thehuman IgG and anti-human IgG can take place. One or more reagents, whichmay be optionally stored in a channel of the device prior to use, canthen flow over this binding pair complex. One of the stored reagents mayinclude a solution of metal colloid (e.g., a gold conjugated antibody)that specifically binds to the antigen to be detected (e.g., human IgG).This metal colloid can provide a catalytic surface for the deposition ofan opaque material, such as a layer of metal (e.g., silver), on asurface of the analysis region. The layer of metal can be formed byusing a two component system: a metal precursor (e.g., a solution ofsilver salts) and a reducing agent (e.g., hydroquinone,chlorohydroquinone, pyrogallol, metol, 4-aminophenol and phenidone),which can optionally be stored in different channels prior to use.

As a positive or negative pressure differential is applied to thesystem, the silver salt and reducing solutions can merge at a channelintersection, where they mix (e.g., due to diffusion) in a channel, andthen flow over the analysis region. Therefore, if antibody-antigenbinding occurs in the analysis region, the flowing of the metalprecursor solution through the region can result in the formation of anopaque layer, such as a silver layer, due to the presence of thecatalytic metal colloid associated with the antibody-antigen complex.The opaque layer may include a substance that interferes with thetransmittance of light at one or more wavelengths. An opaque layer thatis formed in the channel can be detected optically, for example, bymeasuring a reduction in light transmittance through a portion of theanalysis region (e.g., a serpentine channel region) compared to aportion of an area that does not include the antibody or antigen.Alternatively, a signal can be obtained by measuring the variation oflight transmittance as a function of time, as the film is being formedin an analysis region. The opaque layer may provide an increase in assaysensitivity when compared to techniques that do not form an opaquelayer. Additionally, various amplification chemistries that produceoptical signals (e.g., absorbance, fluorescence, glow or flashchemiluminescence, electrochemiluminescence), electrical signals (e.g.,resistance or conductivity of metal structures created by an electrolessprocess) or magnetic signals (e.g., magnetic beads) can be used to allowdetection of a signal by a detector.

Various types of fluids can be used with the cassettes described herein.As described herein, fluids may be introduced into the cassette at firstuse, and/or stored within the cassette prior to first use. Fluidsinclude liquids such as solvents, solutions and suspensions. Fluids alsoinclude gases and mixtures of gases. When multiple fluids are containedin a cassette, the fluids may be separated by another fluid that ispreferably substantially immiscible in each of the first two fluids. Forexample, if a channel contains two different aqueous solutions, aseparation plug of a third fluid may be substantially immiscible in bothof the aqueous solutions. When aqueous solutions are to be keptseparate, substantially immiscible fluids that can be used as separatorsmay include gases such as air or nitrogen, or hydrophobic fluids thatare substantially immiscible with the aqueous fluids. Fluids may also bechosen based at least in part on the fluid's reactivity with adjacentfluids. For example, an inert gas such as nitrogen may be used in someembodiments and may help preserve and/or stabilize any adjacent fluids.An example of an substantially immiscible liquid for separating aqueoussolutions is perfluorodecalin. The choice of a separator fluid may bemade based on other factors as well, including any effect that theseparator fluid may have on the surface tension of the adjacent fluidplugs. It may be preferred to maximize the surface tension within anyfluid plug to promote retention of the fluid plug as a single continuousunit under varying environmental conditions such as vibration, shock andtemperature variations. Separator fluids may also be inert to a reactionsite (e.g., measurement zone) to which the fluids will be supplied. Forexample, if a reaction site includes a biological binding partner, aseparator fluid such as air or nitrogen may have little or no effect onthe binding partner. The use of a gas (e.g., air) as a separator fluidmay also provide room for expansion within a channel of a fluidic deviceshould liquids contained in the device expand or contract due to changessuch as temperature (including freezing) or pressure variations.

As described herein, a cassette may be configured to operate with ananalyzer in some embodiments. For example, the cassette shownillustratively in FIG. 5 may have a cammed surface along a side portionof the cassette. In this particular embodiment, the cammed surfaceincludes a notch 230 formed at one end of the cassette. The other end ofthe cassette includes a curved surface 232. This cammed surface of thecassette may be configured to interact with a sample analyzer such thatthe analyzer can detect the presence of the cassette within the housingof the analyzer and/or position the cassette within the analyzer.

FIG. 7 shows an example of an analyzer 301 that may be configured toreceive a cassette. The analyzer may include a fluid flow source 40(e.g., a pressure-control system) which may be fluidly connected to thechannels 206, 207, 222 (e.g., of FIG. 6) to pressurize the channels tomove the sample and/or other reagents through the channels. Inparticular, the fluid flow source 40 may be configured to move a sampleand/or reagent initially from the substantially U-shaped channel 222into the first channel 206. The fluid flow source 40 may also be used tomove the reagents in the second channel 207 through the substantiallyU-shaped channel 222 and into the first channel 206. After the sampleand reagents pass through the measurement zones 209 and are analyzed,the fluid flow source 40 may be configured to move the fluids into theabsorbent material 217 of the cassette 200. In one embodiment, the fluidflow source is a vacuum system. It should be understood, however, thatother sources of fluid flow such as valves, pumps, and/or othercomponents can be used.

Analyzer 301 may be used in a variety of ways to process and analyze asample placed within the analyzer. In one particular embodiment, once amechanical component configured to interface with the cassette indicatesthat the cassette 20 is properly loaded in the analyzer 301, theidentification reader reads and identifies information associated withthe cassette 20. The analyzer 301 may be configured to compare theinformation to data stored in a control system to ensure that it hascalibration information for this particular sample (such as acalibration curve or expected values for any measurements made during anassay). In the event that the analyzer does not have the propercalibration information, the analyzer may output a request to the userto upload the specific information needed. This information can beuploaded using, for example, the same identification reader which readsthe cassette information. It could also be uploaded using a separateidentification reader or by some other method. The analyzer may also beconfigured to review expiration date information associated with thecassette and cancel the analysis if the expiration date has passed.

In one embodiment, once the analyzer has determined that the cassettemay be analyzed, a fluid flow source such as a vacuum manifold may beconfigured to contact the cassette to ensure a fluid-tight seal around avacuum port and vent ports of the cassette. In one embodiment, anoptical system may take initial measurements to obtain referencereadings. Such reference readings may be taken both with light sources(e.g., 82, 86 of FIG. 7) activated and deactivated.

To initiate movement of the sample, fluid flow source 40 (e.g., a vacuumsystem) may be activated, which may rapidly change the pressure withinthe channel 206, 207 (e.g., reduced to approximately −30 kPa). Thisreduction of pressure within the channel may drive the sample into thechannel 206 and through each of the measurement zones 209A-209D (seeFIG. 6). After the sample reaches the final measurement zone 209D, thesample may continue to flow into the liquid containment region 217.

In one particular embodiment, the microfluidic sample analyzer 301 isused to measure the level of a prostate specific antigen (PSA) in ablood sample. In this embodiment, four measurement zones 209A-209D maybe utilized to analyze the sample. For example, in a first measurementzone, the walls of the channel may be blocked with a blocking protein(such as Bovine Serum Albumin) such that little or no proteins in theblood sample attach to the walls of the measurement zone 209 (except forperhaps some non-specific binding which may be washed off). This firstmeasurement zone may act as a negative control.

In a second measurement zone 209, the walls of the channel 206 may becoated with a predetermined large quantity of a prostate specificantigen (PSA) to act as a high or positive control. As the blood samplepasses through the second measurement zone 209, little or no PSAproteins in the blood may bind to the walls of the channel. Goldconjugated signal antibodies in the sample may be dissolved from insideof the fluidic connector tube 222 or may be flowed from any othersuitable location. These antibodies may not yet be bound to the PSA inthe sample, and thus they may bind to the PSA on the walls of thechannel to act as a high or positive control.

In a third measurement zone 209, the walls of the channel 206 may becoated with a predetermined small quantity of PSA to act as a lowcontrol. As the blood sample flows through this measurement zone 209, noPSA proteins in the sample bind to the wall of the channel. Goldconjugated signal antibodies in the sample may be dissolved from insideof the fluidic connector tube 222 (which are not yet bound to the PSA inthe sample) or may be flowed from any other suitable location, and maybind to the PSA on the walls of the channel to act as a low control.

In a fourth measurement zone 209, the walls of the channel 206 may becoated with the capture antibody, an anti-PSA antibody, which binds to adifferent epitope on the PSA protein than the gold conjugated signalantibody. As the blood sample flows through the fourth measurement zone,PSA proteins in the blood sample may bind to the anti-PSA antibody in away that is proportional to the concentration of these proteins in theblood. Thus, in one embodiment, the first three measurement zones 209may act as controls and the fourth measurement zone 209 may actuallytest the sample.

In some instances, measurements from a region that analyzes the sample(e.g., the fourth measurement zone described above) can be used not onlyto determine the concentration of an analyte in a sample, but also as acontrol as well. For example, a threshold measurement can be establishedat an early phase of amplification. Measurements above this value (orbelow this value) may indicate that the concentration of analyte isoutside the desired range for the assay. This technique may be used toidentify, for example, whether a High Dose Hook Effect is taking placeduring the analysis, i.e., when a very high concentration of analytegives an artificially low reading.

In other embodiments, different numbers of measurement zones can beprovided, and an analysis may optionally include more than onemeasurement zones that actually test the sample. Additional measurementzones can be used to measure additional analytes so that the system canperform multiplex assays simultaneously with a single sample.

In one particular embodiment, it takes approximately eight minutes for a10 microliter blood sample to flow through the four measurement zones209. The start of this analysis may be calculated when the pressurewithin the channel 206 is approximately −30 kPa. During this time, theoptical system 80 is measuring the light transmission for eachmeasurement zone, and in one embodiment, this data may be transmitted toa control system approximately every 0.1 seconds. Using referencevalues, these measurements may be converted using the followingformulas:

Transmission=(l−ld)/(lr−ld)   (1)

Where:

-   -   l=the intensity of transmitted light through a measurement zone        at a given point in time    -   ld=the intensity of transmitted light through a measurement zone        with the light source off

lr=a reference intensity (i.e. the intensity of the transmitted light ata measurement zone with the light source activated, or before the startof an analysis when only air is in the channel

and

Optical Density=−log(Transmission)   (2)

Thus, using these formulas, the optical density in a measurement zone209 may be calculated.

As described herein, a variety of methods can be used to control fluidflow in a cassette, including the use of pumps, vacuums, valves, andother components associated with an analyzer. In some cases, fluidcontrol can also be performed at least in part by one or more componentswithin the cassette, such as by using a valve positioned within thecassette, or the use of specific fluids and channel configurations withthe cassette. In one set of embodiments, control of fluid flow can beachieved based at least in part on the influence of channel geometry andthe viscosity of one or more fluids (which may be stored) inside thecassette.

One method includes flowing a plug of a low viscosity fluid and a plugof a high viscosity fluid in a channel including a flow constrictionregion and a non-constriction region. In one embodiment, the lowviscosity fluid flows at a first flow rate in the channel and the flowrate is not substantially affected by the fluid flowing in the flowconstriction region. When the high viscosity fluid flows from thenon-constriction region to the flow constriction region, the flow ratesof the fluids decrease substantially, since the flow rates, in somesystems, are influenced by the highest viscosity fluid flowing in thesmallest cross-sectional area of the system (e.g., the flow constrictionregion). This causes the low viscosity fluid to flow at a second, slowerflow rate than its original flow rate, e.g., at the same flow rate atwhich the high viscosity fluid flows in the flow constriction region.

For example, one method of controlling fluid flow may involve flowing afirst fluid from a first channel portion to a second channel portion ina microfluidic system, wherein a fluid path defined by the first channelportion has a larger cross-sectional area than a cross-sectional area ofa fluid path defined by the second channel portion, and flowing a secondfluid in a third channel portion in the microfluidic system in fluidcommunication with the first and second channel portions, wherein theviscosity of the first fluid is different than the viscosity of thesecond fluid, and wherein the first and second fluids are substantiallyincompressible. Without stopping the first or second fluids, avolumetric flow rate of the first and second fluids may be decreased bya factor of at least 3, at least 10, at least 20, at least 30, at least40, or at least 50 in the microfluidic system as a result of the firstfluid flowing from the first channel portion to the second channelportion, compared to the absence of flowing the first fluid from thefirst channel portion to the second channel portion. A chemical and/orbiological interaction involving a component of the first or secondfluids may take place at a first measurement zone in fluid communicationwith the channel portions while the first and second fluids are flowingat the decreased flow rate.

Accordingly, by designing microfluidic systems with flow constrictionregions positioned at particular locations and by choosing appropriateviscosities of fluids, a fluid can be made to speed up or slow down atdifferent locations within the system without the use of valves and/orwithout external control. In addition, the length of the channelportions can be chosen to allow a fluid to remain in a particular areaof the system for a certain period of time. Such systems areparticularly useful for performing chemical and/or biological assays, aswell as other applications in which timing of reagents is important.Non-limiting examples of methods and configurations of channels forcontrolling fluid flow are described in more detail in U.S. patentapplication Ser. No. 12/428,372, filed Apr. 22, 2009, published as U.S.Patent Publication No. 2009/0266421, entitled “Flow Control inMicrofluidic Systems”, which is incorporated herein by reference in itsentirety for all purposes.

Any suitable fluid flow source may be used to promote or maintain fluidflow in a microfluidic system or cassette described herein. In somecases, the fluid flow source is part of a microfluidic sample analyzer.A fluid flow source may be configured to pressurize a channel in acassette to move a sample through the channel In one illustrativeembodiment, the fluid flow source is a vacuum system and includes avacuum source or pump, two vacuum reservoirs which may be separated by avacuum regulator and a manifold to provide a fluid connection betweenthe vacuum reservoirs and the cassette. The manifold may also includeone or more fluid connections to one or more ports on the cassette. Forexample, the manifold may provide a fluidic connection between a portand a valve (such as a solenoid valve). Opening and closing this valvemay control where air can enter the cassette, thus serving as a ventvalve in certain embodiments.

As mentioned above, in one embodiment, the vacuum source is a pump, suchas a solenoid operated diaphragm pump. In other embodiments, fluid flowmay be driven/controlled via use of other types of pumps or sources offluid flow. For example, in one embodiment, a syringe pump may be usedto create a vacuum by pulling the syringe plunger in an outwarddirection. In other embodiments, a positive pressure is applied to oneor more inlets of the cassette to provide a source of fluid flow.

In some embodiments, fluid flow takes place while applying asubstantially constant non-zero pressure drop (i.e., AP) across an inletand an outlet of a cassette. In one set of embodiments, an entireanalysis is performed while applying a substantially constant non-zeropressure drop (i.e., AP) across an inlet and an outlet of a cassette. Asubstantially constant non-zero pressure drop can be achieved, forexample, by applying a positive pressure at the inlet or a reducedpressure (e.g., a vacuum) at the outlet. In some cases, a substantiallyconstant non-zero pressure drop is achieved while fluid flow does nottake place predominately by capillary forces and/or without the use ofactuating valves (e.g., without changing a cross-sectional area of achannel of a fluid path of the cassette). In some embodiments, duringessentially the entire analysis conducted in the cassette, asubstantially constant non-zero pressure drop may be present across, forexample, an inlet to a measurement zone (which may be connected to afluidic connector) and an outlet downstream of the measurement zone(e.g., an outlet downstream of a liquid containment region),respectively.

In one embodiment, a vacuum source is configured to pressurize a channelto approximately −60 kPa (approximately ⅔ atmosphere). In anotherembodiment, the vacuum source is configured to pressurize a channel toapproximately −30 kPa. In certain embodiments, a vacuum sources isconfigured to pressurize a channel to, for example, between −100 kPa and−70 kPa, between −70 kPa and −50 kPa, between −50 kPa and −20 kPa, orbetween −20 kPa and −1 kPa.

As mentioned above, in one embodiment, two vacuum reservoirs may beprovided. The pump may be turned on such that the first reservoir may bepressurized to approximately −60 kPa. A regulator positioned between thereservoirs may ensure that the second reservoir may only be pressurizedto a different pressure, for example, approximately −30 kPa. Thisregulator may maintain the pressure of a reservoir at −30 kPa (or atanother suitable pressure) as long as the other reservoir remains at acertain pressure range, e.g., between −60 kPa and −30 kPa. Pressuresensors may monitor the pressure within each reservoir. If the pressurein the first reservoir reaches a set point (for example, approximately−40 kPa), the pump may be actuated to decrease the pressure in the firstreservoir. The second reservoir may be configured to detect any leaks inthe overall vacuum system. Optionally, the vacuum system may include afilter coupled to the reservoirs. A solenoid valve may serve as a ventvalve connected through the manifold to a port.

In certain embodiments, once the cassette is positioned within ananalyzer, a fluid flow source that is a part of the analyzer may becoupled to the cassette to ensure a fluid-tight connection. Forinstance, the cassette may include a port configured to couple a channelof the cassette with the fluid source, and optionally to another channelof the cassette. In one embodiment, seals, or o-rings are positionedaround the port and a linear solenoid may be positioned above theo-rings to press and seal the o-rings against the cassette body. Amanifold adapter may be positioned between the linear solenoid and themanifold, and passive return springs may be provided around the manifoldto urge the manifold away from the cassette body when the solenoid isnot charged. In one embodiment, multiple ports on the cassette mayinterface with the manifold. For example, in addition to a port forinserting and/or removing reagents, the cassette may also include one ormore venting ports and/or mixing ports. The interface between each portand the manifold may be independent (e.g., there may be no fluidicconnection inside the manifold).

In one embodiment, when the fluid flow source is activated, one or morechannels in the cassette may be pressurized (e.g., to approximately −30kPa) which may drive the fluids within the channel (e.g., both fluidsample as well as reagents) toward the outlet. In an embodiment whichincludes a vent port and a mixing port, a vent valve connected to thevent port through a manifold may initially be open which may enable allof the reagents downstream of the mixing port to move toward the outlet,but will not cause reagents upstream of the mixing port to move.Configurations and uses of vent valves are described in more detail inU.S. Patent Apl. Ser. No. 61/263,981, filed Nov. 14, 2009 and entitled,“Fluid Mixing and Delivery in Microfluidic Systems, which isincorporated herein by reference in its entirety for all purposes. Oncethe vent valve is closed, reagents upstream of the mixing port may movetoward a mixing port and then to the outlet. For example, fluids can bestored serially in a channel upstream of the mixing port, and afterclosing a vent valve positioned along the channel, the fluids can flowsequentially towards the channel outlet. In some cases, fluids can bestored in separate, intersecting channels, and after closing a ventvalve the fluids will flow together toward a point of intersection. Thisset of embodiments can be used, for example, to controllably mix thefluids as they flow together. The timing of delivery and the volume offluid delivered can be controlled, for example, by the timing of thevent valve actuation.

Advantageously, vent valves can be operated without constricting thecross-section of the microfluidic channel on which they operate, asmight occur with certain valves in the prior art. Such a mode ofoperation can be effective in preventing leaking across the valve.Moreover, because vent valves can be used, some systems and methodsdescribed herein do not require the use of certain internal valves,which can be problematic due to, for example, their high expense,complexity in fabrication, fragility, limited compatibility with mixedgas and liquid systems, and/or unreliability in microfluidic systems.

It should be understood that while vent valves are described, othertypes of valving mechanisms can be used with the systems and methodsdescribed herein. Non-limiting examples of a valving mechanism which maybe operatively associated with a valve include a diaphragm valve, ballvalve, gate valve, butterfly valve, globe valve, needle valve, pinchvalve, poppet valve, or pinch valve. The valving mechanism may beactuated by any suitable means, including a solenoid, a motor, by hand,by electronic actuation, or by hydraulic/pneumatic pressure.

As previously mentioned, all of the liquids in the cassette (e.g.,sample and reagents) may move into the liquid containment area which mayinclude an absorbent material. In one embodiment, the absorbent materialabsorbs only liquids such that gases may flow out of the cassettethrough the outlet.

A variety of determination (e.g., measuring, quantifying, detecting, andqualifying) techniques may be used, e.g., to analyze a sample componentor other component or condition associated with a microfluidic system orcassette described herein. Determination techniques may includeoptically-based techniques such as light transmission, light absorbance,light scattering, light reflection and visual techniques. Determinationtechniques may also include luminescence techniques such asphotoluminescence (e.g., fluorescence), chemiluminescence,bioluminescence, and/or electrochemiluminescence. In other embodiments,determination techniques may measure conductivity or resistance. Assuch, an analyzer may be configured to include such and other suitabledetection systems.

Different optical detection techniques provide a number of options fordetermining reaction (e.g., assay) results. In some embodiments, themeasurement of transmission or absorbance means that light can bedetected at the same wavelength at which it is emitted from a lightsource. Although the light source can be a narrow band source emittingat a single wavelength it may also may be a broad spectrum source,emitting over a range of wavelengths, as many opaque materials caneffectively block a wide range of wavelengths. In some embodiments, asystem may be operated with a minimum of optical devices (e.g., asimplified optical detector). For instance, the determining device maybe free of a photomultiplier, may be free of a wavelength selector suchas a grating, prism or filter, may be free of a device to direct orcolumnate light such as a columnator, or may be free of magnifyingoptics (e.g., lenses). Elimination or reduction of these features canresult in a less expensive, more robust device.

In one set of embodiments, an optical system is positioned in thehousing of an analyzer. As shown illustratively in FIG. 7, an opticalsystem 80 includes at least a first light source 82 and a detector 84spaced apart from the first light source. The first light source 82 maybe configured to pass light through a first measurement zone of thecassette 20 when the cassette is inserted into the analyzer 301. Thefirst detector 84 may be positioned opposite the first light source 82to detect the amount of light that passes through the first measurementzone of the cassette. In one particular embodiment, the optical systemincludes ten light sources and ten detectors. It should be appreciatedthat in other embodiments, the number of light sources and detectors mayvary as the invention is not so limited. As described herein, thecassette may include a plurality of measurement zones and the cassettemay be positioned within the analyzer such that each measurement zonealigns with a light source and corresponding detector. In someembodiments, the light source includes an optical aperture which mayhelp direct light from the light source to a particular region within ameasurement zone of the cassette.

In one embodiment, the light sources are light emitting diodes (LED's)or laser diodes. For example, an InGaAlP red semiconductor laser diodeemitting at 654 nm may be used. Other light sources can also be used.The light source may be positioned within a nest or housing. The nest orhousing may include a narrow aperture or thin tube that may assist incollimating light. The light sources may be positioned above where thecassette is inserted into the analyzer such that the light source shinesdown onto the top surface of the cassette. Other suitable configurationsof the light source with respect to the cassette are also possible.

It should be appreciated that the wavelength of the light sources mayvary as the invention is not so limited. For example, in one embodiment,the wavelength of the light source is approximately 670 nm, and inanother embodiment, the wavelength of the light source is approximately650 nm. It should be appreciated that in one embodiment, the wavelengthof each light source may be different such that each measurement zone ofthe cassette receives a different light wavelength. In one particularembodiment when measuring hemocrit or hemoglobin, an isobesticwavelength range between approximately 590 nm and approximately 805 nmmay be used for at least one of the measurement zones.

As mentioned, a detector 84 may be spaced apart from and positionedbelow a light source to detect the amount of light that passes throughthe cassette. In one embodiment, one or more of the detectors arephotodetectors (e.g., photodiodes). In certain embodiments, thephotodetector may be any suitable device capable of detecting thetransmission of light that is emitted by the light source. One type ofphotodetector is an optical integrated circuit (IC) including aphotodiode having a peak sensitivity at 700 nm, an amplifier and avoltage regulator. The detector may be positioned within a nest orhousing which may include a narrow aperture or thin tube to ensure thatonly light from the center of the measurement zone is measured at thedetector. As described in more detail below, if the light source ispulse modulated, the photodetector may include a filter to remove theeffect of light that is not at the selected frequency. When multiple andneighboring signals are detected at the same time, the light source usedfor each measurement zone (e.g., detection region) can be modulated at afrequency sufficiently different from that of its neighboring lightsource. In this configuration, the each detector can be configured(e.g., using software) to select for its attributed light source,thereby avoiding interfering light form neighboring optical pairs.

As described herein, a cassette may include a measurement zone whichincludes a meandering channel configured and arranged to align with adetector such that upon alignment, the detector can measure a singlesignal through more than one adjacent segment of the meandering channel.In some embodiments, the detector is able to detect a signal within atleast a portion of the area of the meandering channel and through morethan one segment of the meandering channel such that a first portion ofthe signal, measured from a first segment of the meandering channel, issimilar to a second portion of the signal, measured from a secondsegment of the meandering channel. In such embodiments, because thesignal is present as a part of more than one segment of the meanderingchannel, there is no need for precise alignment between a detector and ameasurement zone.

The positioning of the detector over the measurement zone (e.g., ameandering region) without the need for precision is an advantage, sinceexternal (and possibly, expensive) equipment such as microscopes,lenses, and alignment stages are not required (although they may be usedin certain embodiments). Instead, alignment may be performed by low-costmethods that do not necessarily require an active or separate alignmentstep by the user. For example, in one embodiment, a cassette comprisinga meandering region can be placed in a slot of an analyzer describedherein (e.g., in a cavity having the same or similar shape as thecassette), and the measurement zone can be automatically located in abeam of light of the detector. Possible causes of misalignment causedby, for instance, cassette-to-cassette variations, the exact location ofthe cassette in the slot, and normal usage of the cassette, may benegligible compared to the dimensions of the measurement zone. As aresult, the meandering region can stay within the beam of light anddetection is not interrupted due to these variations.

The detector may detect a signal within all, or a portion, of ameasurement zone (e.g., including a meandering region). In other words,different amounts of the meandering region may be used as an opticaldetection pathway. For instance, the detector may detect a signal withinat least 15% of the measurement zone, at least 20% of the measurementzone, at least 25% of the measurement zone, within at least 50% of themeasurement zone, or within at least 75% of the measurement zone (butless than 100% of the measurement zone). The area in which themeasurement zone is used as an optical detection pathway may also dependon, for instance, the opacity of the material in which the channel isfabricated (e.g., whether all, or, a portion, of the channel istransparent), the amount of a non-transparent material that may cover aportion of the channel (e.g., via use of a protective cover), and/or thesize of the detector and the measurement zone.

In one embodiment, a signal produced by a reaction carried out in thecassette is homogenous over the entire measurement zone (e.g., over anentire meandering channel region). That is, the measurement zone (e.g.,meandering channel region) may allow production and/or detection of asingle, homogenous signal in said region upon carrying out a chemicaland/or biological reaction (e.g., and upon detection by a detector).Prior to carrying out a reaction in the meandering channel region, themeandering channel may include, for example, a single species (andconcentration of species) to be detected/determined. The species may beadsorbed to a surface of the meandering channel. In another embodiment,the signal may be homogeneous over only portions of the meanderingregion, and one or more detectors may detect different signals withineach of the portions. In certain instances, more than one measurementzone can be connected in series and each measurement zone can be used todetect/determine a different species. It should be understood that whilemeandering regions are described, measurement zones that do not includemeandering regions can also be used.

Applicant has recognized that the amount of light transmitted through ameasurement zone of the cassette may be used to determine informationabout not only the sample, but also information about specific processesoccurring in the fluidic system of the cassette (e.g., mixing ofreagents, flow rate, etc.). In some cases, measurement of light througha region can be used as feedback to control fluid flow in the system, asdescribed herein.

In some cases, optical density of a fluid is determined It should berecognized that a clear liquid (such as water) may allow a large amountof light to be transmitted from the light source, through a measurementzone and to a detector. Air within the measurement zone may lead to lesslight transmitted through the measurement zone because more light mayscatter within the channel compared to when a clear liquid is present.When a blood sample is in a measurement zone, a significantly lessamount of light may pass through to the detector due to the lightscattering off of blood cells and also due to absorbance. In oneembodiment, silver associates with a sample component bound to a surfacewithin the measurement zone and as silver builds up within themeasurement zone, less and less light is transmitted through themeasurement zone.

It is recognized that measuring the amount of light that is detected ateach detector enables a user to determine which reagents are in aparticular measurement zone at a particular point in time. It is alsorecognized that by measuring the amount of light that is detected witheach detector, it is possible to measure the amount of silver depositedin each measurement zone. This amount may correspond to the amount ofanalyte captured during a reaction which may thus provide a measure ofthe concentration of the analyte in the sample.

As described herein, Applicant has recognized that an optical system maybe used for a variety of quality control reasons. First, the time ittakes for a sample to reach a measurement zone where the optical systemdetects the light that passes though the measurement zone may be used todetermine whether there is a leak or clog in the system. Also, when thesample is expected to be a certain volume, for example, approximately 10microliters, there is an expected flow time which would be associatedfor the sample to pass through the channels and measurement zones. Ifthe sample falls outside of that expected flow time, it could be anindication that there is not enough sample to conduct the analysisand/or that the wrong type of sample was loaded into the analyzer.Additionally, an expected range of results may be determined based uponthe type of sample (e.g., serum, blood, urine, etc.) and if the sampleis outside of the expected range, it could be an indication of an error.

In one embodiment, an optical system includes a plurality of lightsources and a plurality of corresponding detectors. In one embodiment, afirst light source is adjacent a second light source, where the firstlight source is configured to pass light though a first measurement zoneof a cassette and the second light source is configured to pass lightthrough a second measurement zone of the cassette. In one embodiment,the light sources are configured such that the second light source isnot activated unless the first light source is deactivated. Applicanthas recognized that some light from one light source may spread over toan adjacent detector and may affect the amount of light detected at theadjacent detector. In one set of embodiments, if the adjacent lightsource is activated at the same time as the first light source, thenboth detectors are also measuring the amount of light that passesthrough the first and second measurement zones of the cassette at thesame time, which may lead to inaccurate measurements.

Thus, in one set of embodiments, the plurality of light sources areconfigured to activate sequentially with only one light source activatedat a time. The corresponding detector for the activated light source isthus only detecting the amount of light that passes through thecorresponding measurement zone. In one particular embodiment, the lightsources are configured to each activate for a short period of time(e.g., at least approximately 500, 250, 100, or 50 microseconds, or, insome embodiments, less than or equal to approximately 500, 250, 100, or50 microseconds), and then an adjacent light source is configured toactivate for a similar time frame. Activation for 100 microsecondscorresponds to a rate of 10 kHz. In one embodiment, a multiplexed analogto digital converter is used to pulse the light and measure the amountof light detected at each corresponding detector every 500, 250, 100, or50 microseconds. Pulsing the light in this manner may help to preventstray light passing through one measurement zone to alter the amount oflight detected that passes through an adjacent measurement zone.

Although there may be some benefits associated with pulsing the lightsources in the manner described above, it should be recognized that theinvention is not so limited and that other arrangements may be possible,such as where multiple light sources may be activated at the same time.For example, in one embodiment, light sources that are not directlyadjacent to one another can be activated substantially simultaneously.

In one embodiment, an analyzer includes a temperature regulating systempositioned within the housing which may be configured to regulate thetemperature within the analyzer. For certain sample analysis, the samplemay need to be kept within a certain temperature range. For example, inone embodiment, it is desirable to maintain the temperature within theanalyzer at approximately 37° C. Accordingly, in one embodiment, thetemperature regulating system includes a heater configured to heat thecassette. In one embodiment, the heater is a resistive heater which maybe positioned on the underside of where the cassette is placed in theanalyzer. In one embodiment, the temperature regulating system alsoincludes a thermistor to measure the temperature of the cassette and acontroller circuit may be provided to control the temperature.

In one embodiment, the passive flow of air within the analyzer may actto cool the air within the analyzer if needed. A fan (not shown) mayoptionally be provided in the analyzer to lower the temperature withinthe analyzer. In some embodiments, the temperature regulating system mayinclude Peltier thermoelectric heaters and/or coolers within theanalyzer.

In certain embodiments, an identification system including one or moreidentifiers is used and associated with one or more components ormaterials associated with a cassette and/or analyzer. The “identifiers,”as described in greater detail below, may themselves be “encoded with”information (i.e. carry or contain information, such as by use of aninformation carrying, storing, generating, or conveying device such as aradio frequency identification (RFID) tag or bar code) about thecomponent including the identifier, or may not themselves be encodedwith information about the component, but rather may only be associatedwith information that may be contained in, for example, a database on acomputer or on a computer readable medium (e.g., information about auser, and/or sample to be analyzed). In the latter instance, detectionof such an identifier can trigger retrieval and usage of the associatedinformation from the database.

Identifiers “encoded with” information about a component need notnecessarily be encoded with a complete set of information about thecomponent. For example, in certain embodiments, an identifier may beencoded with information merely sufficient to enable a uniqueidentification of the cassette (e.g. relating to a serial no., part no.,etc.), while additional information relating to the cassette (e.g. type,use (e.g., type of assay), ownership, location, position, connectivity,contents, etc.) may be stored remotely and be only associated with theidentifier.

“Information about” or “information associated with” a cassette,material, or component, etc. is information regarding the identity,positioning, or location of the cassette, material or component or theidentity, positioning, or location of the contents of a cassette,material or component and may additionally include information regardingthe nature, state or composition of the cassette, material, component orcontents. “Information about” or “information associated with” acassette, material or component or its contents can include informationidentifying the cassette, material or component or its contents anddistinguishing the cassette, material, component or its contents fromothers. For example, “information about” or “information associatedwith” a cassette, material or component or its contents may refer toinformation indicating the type or what the cassette, material orcomponent or its contents is, where it is or should be located, how itis or should be positioned, the function or purpose of the cassette,material or component or its contents, how the cassette, material orcomponent or its contents is to be connected with other components ofthe system, the lot number, origin, calibration information, expirationdate, destination, manufacturer or ownership of the cassette, materialor component or its contents, the type of analysis/assay to be performedin the cassette, information about whether the cassette has beenused/analyzed, etc.

In one set of embodiments, an identifier is associated with a cassetteand/or analyzer described herein. In general, as used herein, the term“identifier” refers to an item capable of providing information aboutthe cassette and/or analyzer (e.g. information including one or more ofidentity, location, or position/positioning of the cassette and/oranalyzer or a component thereof) with which the identifier is associatedor installed into, or capable of being identified or detected and theidentification or detection event being associated with informationabout the cassette and/or analyzer with which the identifier isassociated. Non-limiting examples of identifiers that may be used in thecontext of the invention include radio frequency identification (RFID)tags, bar codes, serial numbers, color tags, fluorescent or optical tags(e.g., using quantum dots), chemical compounds, radio tags, magnetictags, among others.

In one embodiment, an analyzer may include an identification readerpositioned within the housing configured to read information about withthe cassette. Any suitable identification reader that can be used toread information from an identifier. Non-limiting examples ofidentification readers include RFID readers, bar code scanners, chemicaldetectors, cameras, radiation detectors, magnetic or electric fielddetectors, among others. The method of detection/reading and appropriatetype of identification detector depends on the particular identifierutilized and can include, for example, optical imaging, fluorescenceexcitation and detection, mass spectrometry, nuclear magnetic resonance,sequencing, hybridization, electrophoresis, spectroscopy, microscopy,etc. In some embodiments, the identification readers may be mounted orpre-embedded in specific locations (e.g., on or within a cassette and/oranalyzer).

In one embodiment, the identification reader is an RFID readerconfigured to read an RFID identifier associated with the cassette. Forexample, in one embodiment, the analyzer includes an RFID module andantenna that are configured to read information from the cassetteinserted into the analyzer. In another embodiment, the identificationreader is a barcode reader configured to read a barcode associated withthe cassette. Once the cassette is inserted into the analyzer, theidentification reader may read the information from the cassette. Theidentifier on the cassette may include one or more of the types ofinformation such as cassette type, type of analysis/assay to beperformed, lot number, information about whether the cassette has beenused/analyzed, and other information described herein. The reader mayalso be configured to read information provided with a group ofcassettes, such as in a box of cassettes, such as, but not limited tocalibration information, expiration date, and any additional informationspecific to that lot. The information identified may be optionallydisplayed to a user, e.g., to confirm that a correct cassette and/ortype of assay is being performed.

In some cases, the identification reader may be integrated with acontrol system via communication pathways. Communication between theidentification readers and the control system may occur along ahard-wired network or may be transmitted wirelessly. In one embodiment,the control system can be programmed to recognize a specific identifier(e.g., of a cassette associated with information relating to a cassettetype, manufacturer, assay to be performed, etc.) as indicating thecassette is suitably connected or inserted within a particular type ofanalyzer.

In one embodiment, the identifier of a cassette be associated withpredetermined or programmed information contained in a databaseregarding the use of the system or cassette for a particular purpose,user or product, or with particular reaction conditions, sample types,reagents, users, and the like. If an incorrect match is detected or anidentifier has been deactivated, the process may be halted or the systemmay be rendered not operable until the user has been notified, or uponacknowledgement by a user.

The information from or associated with an identifier can, in someembodiments, be stored, for example in computer memory or on a computerreadable medium, for future reference and record-keeping purposes. Forexample, certain control systems may employ information from orassociated with identifiers to identify which components (e.g.,cassettes) or type of cassettes were used in a particular analysis, thedate, time, and duration of use, the conditions of use, etc. Suchinformation may be used, for example, to determine whether one or morecomponents of the analyzer should be cleaned or replaced. Optionally, acontrol system or any other suitable system could generate a report fromgathered information, including information encoded by or associatedwith the identifiers, that may be used in providing proof of compliancewith regulatory standards or verification of quality control.

Information encoded on or associated with an identifier may also beused, for example, to determine whether the component associated withthe identifier (e.g., a cassette) is authentic or counterfeit. In someembodiments, the determination of the presence of a counterfeitcomponent causes system lockout. In one example, the identifier maycontain a unique identity code. In this example, the process controlsoftware or analyzer would not permit system startup (e.g., the systemmay be disabled) if a foreign or mismatched identity code (or noidentity code) was detected.

In certain embodiments, the information obtained from or associated withan identifier can be used to verify the identity of a customer to whomthe cassette and/or analyzer is sold or for whom a biological, chemical,or pharmaceutical process is to be performed. In some cases, theinformation obtained from or associated with an identifier is used aspart of a process of gathering data for troubleshooting a system. Theidentifier may also contain or be associated with information such asbatch histories, assembly process and instrumentation diagrams (P andIDs), troubleshooting histories, among others. Troubleshooting a systemmay be accomplished, in some cases, via remote access or include the useof diagnostic software.

In one embodiment, an analyzer includes a user interface, which may bepositioned within the housing and configured for a user to inputinformation into the sample analyzer. In one embodiment, the userinterface includes a touch screen. The touch screen may guide a userthrough the operation of the analyzer, providing text and/or graphicalinstructions for use of the analyzer. The user interface may guide theuser to input the patient's name or other patient identificationsource/number into the analyzer. Any suitable patient information suchas name, date of birth, and/or patient ID number may be inputted intothe touch screen user interface to identify the patient. The userinterface may indicate the amount of time remaining to complete theanalysis of the sample.

In another embodiment, the user interface may be configured differently,such as with an LCD display and a single button scroll through menu. Inanother embodiment, the user interface may simply include a start buttonto activate the analyzer. In other embodiments, the user interface fromseparate independent devices (such as a smart phone or mobile computer)can be used to interface with the analyzer. FIG. 8 is a block diagram300 that illustrates how a control system 305 (see FIG. 7) may beoperatively associated with a variety of different components accordingto one embodiment. Control systems described herein can be implementedin numerous ways, such as with dedicated hardware or firmware, using aprocessor that is programmed using microcode or software to perform thefunctions recited above or any suitable combination of the foregoing. Acontrol system may control one or more operations of a single analysis(e.g., for a biological, biochemical or chemical reaction), or ofmultiple (separate or interconnected) analyses. As shown illustrativelyin FIG. 7, control system 305 may be positioned within the housing 101of the analyzer and may be configured to communicate with theidentification reader 60, the user interface 200, the fluid flow source40, the optical system 80, and/or the temperature regulating system toanalyze a sample in the cassette.

In one embodiment, the control system includes at least two processors,including a real time processor that controls and monitors all of thesub-systems which directly interface with the cassette. In oneembodiment, at a particular time interval (e.g., every 0.1 seconds),this processor communicates with a second higher level processor whichcommunicates with the user through the user interface and/or thecommunication sub-system (discussed below) and directs the operation ofthe analyzer (e.g., determines when to start analyzing a sample andinterprets the results). In one embodiment, communication between thesetwo processors occurs through a serial communication bus. It should beappreciated that in another embodiment, the analyzer may only includeone processor, or more than two processors, as the invention is not solimited.

In one embodiment, the analyzer is capable of interfacing with externaldevices and may, for example, include ports for connection with one ormore external communication units. External communication may beaccomplished, for example, via USB communication. For example, as shownillustratively in FIG. 8, the analyzer may output the results of asample analysis to a USB printer 400, or to a computer 402.Additionally, the data stream produced by the real time processor may beoutputted to a computer or a USB memory stick 404. In some embodiments,a computer may be able to directly control the analyzer through a USBconnection as well. Further, other types of communication options areavailable as the present invention is not limited in this respect. Forexample, Ethernet, Bluetooth and/or WI-FI communication 406 with theanalyzer may be established through the processor.

The calculation methods, steps, simulations, algorithms, systems, andsystem elements described herein may be implemented using a computerimplemented control system, such as the various embodiments of computerimplemented systems described below. The methods, steps, systems, andsystem elements described herein are not limited in their implementationto any specific computer system described herein, as many otherdifferent machines may be used.

The computer implemented control system can be part of or coupled inoperative association with a sample analyzer, and, in some embodiments,configured and/or programmed to control and adjust operationalparameters of the sample analyzer, as well as analyze and calculatevalues, as described above. In some embodiments, the computerimplemented control system can send and receive reference signals to setand/or control operating parameters of the sample analyzer and,optionally, other system apparatus. In other embodiments, the computerimplemented system can be separate from and/or remotely located withrespect to the sample analyzer and may be configured to receive datafrom one or more remote sample analyzer apparatus via indirect and/orportable means, such as via portable electronic data storage devices,such as magnetic disks, or via communication over a computer network,such as the Internet or a local intranet.

A computer implemented control system may include several knowncomponents and circuitry, including a processing unit (i.e., processor),a memory system, input and output devices and interfaces (e.g., aninterconnection mechanism), as well as other components, such astransport circuitry (e.g., one or more busses), a video and audio datainput/output (I/O) subsystem, special-purpose hardware, as well as othercomponents and circuitry, as described below in more detail. Further,the computer system may be a multi-processor computer system or mayinclude multiple computers connected over a computer network.

The computer implemented control system may include a processor, forexample, a commercially available processor such as one of the seriesx86, Celeron and Pentium processors, available from Intel, similardevices from AMD and Cyrix, the 680X0 series microprocessors availablefrom Motorola, and the PowerPC microprocessor from IBM. Many otherprocessors are available, and the computer system is not limited to aparticular processor.

A processor typically executes a program called an operating system, ofwhich WindowsNT, Windows 95 or 98, UNIX, Linux, DOS, VMS, MacOS and OS8are examples, which controls the execution of other computer programsand provides scheduling, debugging, input/output control, accounting,compilation, storage assignment, data management and memory management,communication control and related services. The processor and operatingsystem together define a computer platform for which applicationprograms in high-level programming languages are written. The computerimplemented control system is not limited to a particular computerplatform.

The computer implemented control system may include a memory system,which typically includes a computer readable and writeable non-volatilerecording medium, of which a magnetic disk, optical disk, a flash memoryand tape are examples. Such a recording medium may be removable, forexample, a floppy disk, read/write CD or memory stick, or may bepermanent, for example, a hard drive.

Such a recording medium stores signals, typically in binary form (i.e.,a form interpreted as a sequence of one and zeros). A disk (e.g.,magnetic or optical) has a number of tracks, on which such signals maybe stored, typically in binary form, i.e., a form interpreted as asequence of ones and zeros. Such signals may define a software program,e.g., an application program, to be executed by the microprocessor, orinformation to be processed by the application program.

The memory system of the computer implemented control system also mayinclude an integrated circuit memory element, which typically is avolatile, random access memory such as a dynamic random access memory(DRAM) or static memory (SRAM). Typically, in operation, the processorcauses programs and data to be read from the non-volatile recordingmedium into the integrated circuit memory element, which typicallyallows for faster access to the program instructions and data by theprocessor than does the non-volatile recording medium.

The processor generally manipulates the data within the integratedcircuit memory element in accordance with the program instructions andthen copies the manipulated data to the non-volatile recording mediumafter processing is completed. A variety of mechanisms are known formanaging data movement between the non-volatile recording medium and theintegrated circuit memory element, and the computer implemented controlsystem that implements the methods, steps, systems and system elementsdescribed above in relation to FIG. 8 is not limited thereto. Thecomputer implemented control system is not limited to a particularmemory system.

At least part of such a memory system described above may be used tostore one or more data structures (e.g., look-up tables) or equationsdescribed above. For example, at least part of the non-volatilerecording medium may store at least part of a database that includes oneor more of such data structures. Such a database may be any of a varietyof types of databases, for example, a file system including one or moreflat-file data structures where data is organized into data unitsseparated by delimiters, a relational database where data is organizedinto data units stored in tables, an object-oriented database where datais organized into data units stored as objects, another type ofdatabase, or any combination thereof.

The computer implemented control system may include a video and audiodata I/O subsystem. An audio portion of the subsystem may include ananalog-to-digital (A/D) converter, which receives analog audioinformation and converts it to digital information. The digitalinformation may be compressed using known compression systems forstorage on the hard disk to use at another time. A typical video portionof the I/O subsystem may include a video image compressor/decompressorof which many are known in the art. Such compressor/decompressorsconvert analog video information into compressed digital information,and vice-versa. The compressed digital information may be stored on harddisk for use at a later time.

The computer implemented control system may include one or more outputdevices. Example output devices include a cathode ray tube (CRT)display, liquid crystal displays (LCD) and other video output devices,printers, communication devices such as a modem or network interface,storage devices such as disk or tape, and audio output devices such as aspeaker.

The computer implemented control system also may include one or moreinput devices. Example input devices include a keyboard, keypad, trackball, mouse, pen and tablet, communication devices such as describedabove, and data input devices such as audio and video capture devicesand sensors. The computer implemented control system is not limited tothe particular input or output devices described herein.

It should be appreciated that one or more of any type of computerimplemented control system may be used to implement various embodimentsdescribed herein. Aspects of the invention may be implemented insoftware, hardware or firmware, or any combination thereof. The computerimplemented control system may include specially programmed, specialpurpose hardware, for example, an application-specific integratedcircuit (ASIC). Such special-purpose hardware may be configured toimplement one or more of the methods, steps, simulations, algorithms,systems, and system elements described above as part of the computerimplemented control system described above or as an independentcomponent.

The computer implemented control system and components thereof may beprogrammable using any of a variety of one or more suitable computerprogramming languages. Such languages may include procedural programminglanguages, for example, C, Pascal, Fortran and BASIC, object-orientedlanguages, for example, C++, Java and Eiffel and other languages, suchas a scripting language or even assembly language.

The methods, steps, simulations, algorithms, systems, and systemelements may be implemented using any of a variety of suitableprogramming languages, including procedural programming languages,object-oriented programming languages, other languages and combinationsthereof, which may be executed by such a computer system. Such methods,steps, simulations, algorithms, systems, and system elements can beimplemented as separate modules of a computer program, or can beimplemented individually as separate computer programs. Such modules andprograms can be executed on separate computers.

Such methods, steps, simulations, algorithms, systems, and systemelements, either individually or in combination, may be implemented as acomputer program product tangibly embodied as computer-readable signalson a computer-readable medium, for example, a non-volatile recordingmedium, an integrated circuit memory element, or a combination thereof.For each such method, step, simulation, algorithm, system, or systemelement, such a computer program product may comprise computer-readablesignals tangibly embodied on the computer-readable medium that defineinstructions, for example, as part of one or more programs, that, as aresult of being executed by a computer, instruct the computer to performthe method, step, simulation, algorithm, system, or system element.

It should be appreciated that various embodiments may be formed with oneor more of the above-described features. The above aspects and featuresmay be employed in any suitable combination as the present invention isnot limited in this respect. It should also be appreciated that thedrawings illustrate various components and features which may beincorporated into various embodiments. For simplification, some of thedrawings may illustrate more than one optional feature or component.However, the invention is not limited to the specific embodimentsdisclosed in the drawings. It should be recognized that the inventionencompasses embodiments which may include only a portion of thecomponents illustrated in any one drawing figure, and/or may alsoencompass embodiments combining components illustrated in multipledifferent drawing figures.

EXAMPLES

The following example is intended to illustrate certain embodiments ofthe present invention, but do not exemplify the full scope of theinvention.

Example 1

This example describes the use of a cassette and analyzer to perform anassay to detect PSA in a sample by electrolessly depositing silver ontogold particles that are associated with the sample. FIG. 9 includes aschematic illustration of a microfluidic system 500 of a cassette usedin this example. The cassette had a similar shape to cassette 20 shownin FIG. 3. The microfluidic system used in this example is generallydescribed in International Patent Publication No. WO2005/066613(International Patent Application Serial No. PCT/US2004/043585), filedDec. 20, 2004 and entitled “Assay Device and Method,” which isincorporated herein by reference in its entirety for all purposes.

The microfluidic system included measurement zones 510A-510D, wastecontainment region 512, and an outlet 514. The measurement zonesincluded a microfluidic channel 50 microns deep and 120 microns wide,with a total length of 175 mm The microfluidic system also includedmicrofluidic channel 516 and channel branches 518 and 520 (with inlets519 and 521, respectively). Channel branches 518 and 520 were 350microns deep and 500 microns wide. Channel 516 was formed ofsub-channels 515, which were 350 microns deep and 500 microns widelocated on alternating sides of the cassette, connected by through holes517 having a diameter of approximately 500 microns. Although FIG. 9shows that reagents were stored on a single side of the cassette, inother embodiments, reagents were stored on both sides of the cassette.Channel 516 had a total length of 390 mm, and branches 518 and 520 wereeach 360 mm long. Before sealing the channels, anti-PSA antibodies wereattached to a surface of the microfluidic system in a segment of themeasurement zone 510.

Prior to first use, the microfluidic system was loaded with liquidreagents which were stored in the cassette. A series of 7 wash plugs523-529 (either water of buffer, approximately 2 microliters each) wereloaded using a pipette into sub-channels 515 of channel 516 using thethru-holes. Each of the wash plugs was separated by plugs of air. Fluid528, containing a solution of silver salt, was loaded into branchingchannel through port 519 using a pipette. Fluid 530, containing areducing solution, was loaded into branching channel 520 through port521. Each of the liquids shown in FIG.9 were separated from the otherliquids by plugs of air. Ports 514, 519, 521, 536, 539, and 540 weresealed with an adhesive tape that can be easily removed or pierced. Assuch, the liquids were stored in the microfluidic system prior to firstuse.

At first use, the ports 514, 519, 521, 536, 539, and 540 were unsealedby a user peeling off a tape covering the opening of the ports. A tube544 containing lyophilized anti-PSA antibodies labeled with colloidalgold and to which 10 microliters of sample blood (522) was added, wasconnected to ports 539 and 540. The tube was part of a fluid connectorhaving a shape and configuration shown in FIG. 3. This created a fluidicconnection between measurement zone 510 and channel 516, which wereotherwise unconnected and not in fluid communication with one anotherprior to first use.

The cassette including microfluidic system 500 was inserted into anopening of an analyzer (e.g., as shown in FIG. 7). The housing of theanalyzer included an arm positioned within the housing that wasconfigured to engage a cammed surface on the cassette. The arm extendedat least partially into the opening in the housing such that as thecassette was inserted into the opening, the arm was pushed away from theopening into a second position allowing the cassette to enter theopening. Once the arm engaged the inwardly cammed surface of thecassette, the cassette was positioned and retained within the housing ofthe analyzer, and the bias of the spring prevented the cassette fromslipping out of the analyzer. The analyzer senses the cassette'sinsertion by means of a position sensor.

An identification reader (RFID reader) positioned within the housing ofthe analyzer was used to read an RFID tag on the cassette which includeslot identification information. The analyzer used this identifier tomatch lot information (e.g., calibration information, expiration date ofthe cassette, verification that the cassette is new, and the type ofanalysis/assay to be performed in the cassette) stored in the analyzer.The user was prompted to input information about the patient (from whichthe sample was acquired) into the analyzer using the touch screen. Afterthe information about the cassette was verified by the user, the controlsystem initiated the analysis.

The control system included programmed instructions to perform theanalysis. To initiate the analysis, a signal was sent to the electronicscontrolling a vacuum system, which was a part of the analyzer and usedto provide fluid flow. A manifold with o-rings was pressed against thecassette surface by a solenoid. One port on the manifold sealed (by ano-ring) to port 536 of the microfluidic system of the cassette. Thisport on the manifold was connected by a tube to a simple solenoid valve(SMC V124A-6G-M5, not shown) which was open to the atmosphere. Aseparate vacuum port on the manifold sealed (by-o-ring) to port 514 ofthe microfluidic system of the cassette. A vacuum of approximately −30kPa was applied to port 514. Throughout the analysis, the channelincluding measurement zone 510 positioned between ports 540 and 514 hada substantially constant non-zero pressure drop of approximately −30kPa. Sample 522 was flowed in the direction of arrow 538 into each ofmeasurement zones 510A-510D. As the fluid passed through the measurementzones, the PSA proteins in sample 522 were captured by anti-PSAantibodies immobilized on the measurement zone walls, as described inmore detail below. The sample took about 7-8 minutes to pass through themeasurement zone, after which it was captured in the waste containmentregion 512.

Initiation of the analysis also involved the control system sending asignal to the optical detectors, which were positioned adjacent each ofmeasurement zones 510, to initiate detection. Each of the detectorsassociated with the measurement zones recorded the transmission of lightthrough the channels of the measurement zones, as shown in a plot 600illustrated in FIG. 10. As the sample passed by each of the measurementzones, peaks 610A-610D were produced. The peaks (and troughs) measuredby the detectors are signals (or are converted to signals) that are sentto the control system which compared the measured signals to referencesignals or values pre-programmed into the control system. The controlsystem included a pre-programmed set of instructions for providingfeedback to the microfluidic system based at least in part on thecomparison of signals/values.

In a first measurement zone 510-A of device 500 of FIG. 9, the walls ofthe channel of this measurement zone were blocked with a blockingprotein (Bovine Serum Albumin) prior to first use (e.g., prior tosealing the device). Little or no proteins in the blood sample attachedto the walls of the measurement zone 510-A (except for perhaps somenon-specific binding which may be washed off). This first measurementzone acted as a negative control.

In a second measurement zone 510-B, the walls of the channel of thismeasurement zone were coated with a predetermined large quantity of aprostate specific antigen (PSA) prior to first use (e.g., prior tosealing the device) to act as a high or positive control. As the bloodsample passed through the second measurement zone 510-B, little or noPSA proteins in the blood bound to the walls of the channel. Goldconjugated signal antibodies in the sample may not yet be bound to thePSA in the sample, and thus they may bind to the PSA on the walls of thechannel to act as a high or positive control.

In a third measurement zone 510-C, the walls of the channel of thismeasurement zone were coated with a predetermined low quantity of PSAprior to first use (e.g., prior to sealing the device) to act as a lowcontrol. As the blood sample flowed through this measurement zone,little or no PSA proteins in the sample bind to the wall of the channelGold conjugated signal antibodies in the sample may bind to the PSA onthe walls of the channel to act as a low control.

In a fourth measurement zone 510-D, the walls of the channel of thismeasurement zone were coated with the capture antibody, an anti-PSAantibody, which binds to a different epitope on the PSA protein than thegold conjugated signal antibody. The walls were coated prior to firstuse (e.g., prior to sealing the device). As the blood sample flowedthrough the fourth measurement zone during use, PSA proteins in theblood sample bound to the anti-PSA antibody in a way that isproportional to the concentration of these proteins in the blood. Sincethe sample, which included PSA, also included gold-labeled anti-PSAantibodies coupled to the PSA, the PSA captured on the measurement zonewalls formed a sandwich immunocomplex.

Wash fluids 523-529 followed the sample through the measurement zones510 towards waste containment region 512 in the direction of arrow 538.As the wash fluids were passed through the measurement zones, theywashed away remaining unbound sample components. Each wash plug cleanedthe channels of the measurement zones, providing progressively morecomplete cleaning. The last wash fluid 529 (water) washed away saltsthat could react with silver salts (e.g., chloride, phosphate, azide).

As shown in the plot illustrated in FIG. 10, while the wash fluids wereflowing through the measurement zones, each of the detectors associatedwith the measurement zones measures a pattern 620 of peaks and troughs.The troughs corresponded to the wash plugs (which are clear liquids andthus provide maximum light transmission). The peaks between each plugrepresent the air between each plug of clear liquid. Since the assayincluded 7 wash plugs, 7 troughs and 7 peaks are present in plot 600.The first trough 622 is generally not as deep as the other troughs 624since the first wash plug often catches blood cells left in the channeland thus is not completely clear.

The final peak of air 628 is much longer than the previous peaks becausethere were no wash plugs to follow. As a detector detects the length ofthis air peak, one or more signals is sent to the control system whichcompares the length of time of this peak to a pre-set reference signalor input value having a particular length. If the length of time of themeasured peak is long enough compared to the reference signal, thecontrol system sends a signal to the electronics controlling vent valve536 to actuate the valve and initiate mixing of fluids 528 and 530.(Note that the signal of peak of air 628 may be combined with a signalindicating either 1) the intensity of the peak; 2) where this peak ispositioned as a function of time, and/or 3) one or more signalsindicating that a series of peaks 620 of particular intensity hasalready passed. In this way, the control system distinguishes peak ofair 628 from other peaks of long duration such as peak 610 from thesample, e.g., using a pattern of signals.)

To initiate mixing, the solenoid connected by the manifold to vent port536 is closed. Since the vacuum remains on and no air can enter throughvent valve 536, air enters the device through ports 519 and 521 (whichare open). This forces the two fluids 528 and 530 in the two storagechannels upstream of vent valve 536 to move substantially simultaneouslytoward outlet 514. These reagents mix at the intersection of thechannels to form an amplification reagent (a reactive silver solution)having a viscosity of about 1×10⁻³ Pa·s. The ratio of the volumes offluids 528 and 530 was about 1:1. The amplification reagent continuedthrough the downstream storage channel, through tube 544, throughmeasurement zones 510, and then to waste containment region 512. After aset amount of time (12 seconds), the analyzer reopened vent valve 536such that air flows through vent valve 536 (instead of the vent ports).This left some reagent behind in the upstream storage channels 518 and520 on the device. This also results in a single plug of mixedamplification reagent. The 12 seconds of vent-valve closure results inan amplification plug of approximately 50 μL. (Instead of simple timing,another way to trigger the re-opening of the vent valve would be todetect the amplification reagent as it first enters the measurementzones.)

Because the mixed amplification reagent is stable for only a few minutes(usually less than 10 minutes), the mixing was performed less than aminute before use in measurement zone 510. The amplification reagent isa clear liquid, so when it enters the measurement zones, optical densityis at its lowest. As the amplification reagent passed across themeasurement zones, silver was deposited on the captured gold particlesto increase the size of the colloids to amplify the signal. (As notedabove, gold particles were present in the low and high positive controlmeasurement zones and, to the extent that PSA was present in the sample,in the test measurement zone.) Silver can then be deposited on top ofthe already deposited silver, leaving more and more silver deposited inthe measurement zones. Eventually the deposited silver reduces thetransmission of light through the measurement zones. The reduction intransmitted light is proportional to the amount of silver deposited andcan be related to the amount of gold colloids captured on the channelwalls. In a measurement zone where no silver is deposited (the negativecontrol for example, or the test area when the sample contains none ofthe target protein, such as PSA), there will be no (or minimal) increasein optical density. In a measurement zone with significant silverdeposition, the slope and ultimate level of the pattern of increasingoptical density will be high. The analyzer monitors the pattern of thisoptical density during amplification in the test area to determine theconcentration of analyte in the sample. In one version of the test, thepattern is monitored within the first three minutes of amplification.The optical density in each of the measurement zones as a function oftime was recorded and are shown as curves 640, 644, 642, and 646 in FIG.10. These curves corresponded to signals that were produced inmeasurement zones 510-A, 510-B, 510-C, and 510-D, respectively.

After three minutes of amplification, the analyzer stops the test. Nomore optical measurements are recorded and the manifold is disengagedfrom the device. The test result is displayed on the analyzer screen andcommunicated to a printer, computer, or whatever output the user hasselected. The user may remove the device from the analyzer and throw itaway. The sample and all the reagents used in the assay remain in thedevice. The analyzer is ready for another test.

It should be noted that the control of the flow rates of the fluidswithin channel 516 and the measurement zone 510 were important whenflowing fluids through the system. Due to the measurement zone'srelatively small cross sectional area, it served as a bottleneck,controlling the overall flow rate in the system. When the measurementzone contained liquids, the linear flow rates of the fluids in channel516 was about 0.5 mm s⁻¹. Fluids flowing from branching channels 518 and520 into main channel 516 might not have mixed reproducibly at thisrate, as one fluid might have flowed faster than the other, causingunequal portions of fluids 528 and 530 to be mixed. On the other hand,when the measurement zone contained air, the linear flow rates of thefluids in channel 516 and branching channels 518 and 520 were about 15mm s⁻¹. At this higher flow rate, the flow rate in branching channels518 and 520 were equal and reproducible (when vent valve 536 wasclosed), producing reproducible mixing. For this reason, the valveconnected to port 536 was not closed until fluid 542 passed through themeasurement zone to the waste containment region. As noted above,determination of when fluid 542 had exited the measurement zone 510 wasperformed using an optical detector so as to measure transmission oflight through part of measurement zone 510 in combination with afeedback system.

The microfluidic system shown in FIG. 9 was designed such that thevolume of the channel between vent valve 536 and measurement zone 510was larger than the expected volume of the mixed activated silversolution (i.e., the combined portion of fluids 528 and 530 whichtraveled into channel 516 while vent valve 536 was closed). This ensuredthat substantially all of the mixing took place at a relatively highlinear flow rate (since no liquid, and only air, was present in themeasurement zone 510 at this time), and before the activated solutionreached the measurement zone. This configuration helped promotereproducible and equal mixing. For the assay described in this example,it was important to sustain a flow of the activated silver mixturewithin the measurement zone for a few minutes (e.g., 2 to 10 minutes).

This example shows that analysis of a sample in a microfluidic system ofa cassette can be performed by using an analyzer that controls fluidflow in the cassette, and by using feedback from one or more measuredsignals to modulate fluid flow.

While several embodiments of the present invention have been describedand illustrated herein, those of ordinary skill in the art will readilyenvision a variety of other means and/or structures for performing thefunctions and/or obtaining the results and/or one or more of theadvantages described herein, and each of such variations and/ormodifications is deemed to be within the scope of the present invention.More generally, those skilled in the art will readily appreciate thatall parameters, dimensions, materials, and configurations describedherein are meant to be exemplary and that the actual parameters,dimensions, materials, and/or configurations will depend upon thespecific application or applications for which the teachings of thepresent invention is/are used. Those skilled in the art will recognize,or be able to ascertain using no more than routine experimentation, manyequivalents to the specific embodiments of the invention describedherein. It is, therefore, to be understood that the foregoingembodiments are presented by way of example only and that, within thescope of the appended claims and equivalents thereto, the invention maybe practiced otherwise than as specifically described and claimed. Thepresent invention is directed to each individual feature, system,article, material, kit, and/or method described herein. In addition, anycombination of two or more such features, systems, articles, materials,kits, and/or methods, if such features, systems, articles, materials,kits, and/or methods are not mutually inconsistent, is included withinthe scope of the present invention.

The indefinite articles “a” and “an,” as used herein in thespecification and in the claims, unless clearly indicated to thecontrary, should be understood to mean “at least one.”

The phrase “and/or,” as used herein in the specification and in theclaims, should be understood to mean “either or both” of the elements soconjoined, i.e., elements that are conjunctively present in some casesand disjunctively present in other cases. Other elements may optionallybe present other than the elements specifically identified by the“and/or” clause, whether related or unrelated to those elementsspecifically identified unless clearly indicated to the contrary. Thus,as a non-limiting example, a reference to “A and/or B,” when used inconjunction with open-ended language such as “comprising” can refer, inone embodiment, to A without B (optionally including elements other thanB); in another embodiment, to B without A (optionally including elementsother than A); in yet another embodiment, to both A and B (optionallyincluding other elements); etc.

As used herein in the specification and in the claims, “or” should beunderstood to have the same meaning as “and/or” as defined above. Forexample, when separating items in a list, “or” or “and/or” shall beinterpreted as being inclusive, i.e., the inclusion of at least one, butalso including more than one, of a number or list of elements, and,optionally, additional unlisted items. Only terms clearly indicated tothe contrary, such as “only one of or “exactly one of,” or, when used inthe claims, “consisting of,” will refer to the inclusion of exactly oneelement of a number or list of elements. In general, the term “or” asused herein shall only be interpreted as indicating exclusivealternatives (i.e. “one or the other but not both”) when preceded byterms of exclusivity, such as “either,” “one of,” “only one of,” or“exactly one of.” “Consisting essentially of,” when used in the claims,shall have its ordinary meaning as used in the field of patent law.

As used herein in the specification and in the claims, the phrase “atleast one,” in reference to a list of one or more elements, should beunderstood to mean at least one element selected from any one or more ofthe elements in the list of elements, but not necessarily including atleast one of each and every element specifically listed within the listof elements and not excluding any combinations of elements in the listof elements. This definition also allows that elements may optionally bepresent other than the elements specifically identified within the listof elements to which the phrase “at least one” refers, whether relatedor unrelated to those elements specifically identified. Thus, as anon-limiting example, “at least one of A and B” (or, equivalently, “atleast one of A or B,” or, equivalently “at least one of A and/or B”) canrefer, in one embodiment, to at least one, optionally including morethan one, A, with no B present (and optionally including elements otherthan B); in another embodiment, to at least one, optionally includingmore than one, B, with no A present (and optionally including elementsother than A); in yet another embodiment, to at least one, optionallyincluding more than one, A, and at least one, optionally including morethan one, B (and optionally including other elements); etc.

In the claims, as well as in the specification above, all transitionalphrases such as “comprising,” “including,” “carrying,” “having,”“containing,” “involving,” “holding,” and the like are to be understoodto be open-ended, i.e., to mean including but not limited to. Only thetransitional phrases “consisting of and “consisting essentially of shallbe closed or semi-closed transitional phrases, respectively, as setforth in the United States Patent Office Manual of Patent ExaminingProcedures, Section 2111.03.

1. A method comprising: initiating detection of fluids at a firstmeasurement zone of a microfluidic system; detecting a first fluid and asecond fluid at the first measurement zone and forming a first signalcorresponding to the first fluid and a second signal corresponding tothe second fluid; transmitting a first pattern of signals to a controlsystem, the first pattern of signals comprising at least two of: a) anintensity of the first signal; b) a duration of the first signal; c) aposition of the first signal in time relative to a second position intime; and d) an average time period between the first and secondsignals; and determining whether to modulate fluid flow in themicrofluidic system based at least in part on the first pattern ofsignals.
 2. A method comprising: detecting a first fluid and a secondfluid at a first measurement zone of a microfluidic system, wherein thedetection step comprises detecting at least two of: a) an opacity of thefirst fluid; b) a volume of the first fluid; c) a flow rate of the firstfluid; d) a position of the detection of the first fluid in timerelative to a second position in time; and e) an average time periodbetween the detection of the first and second fluids; and determiningwhether to modulate fluid flow in the microfluidic system based at leastin part on the detection step.
 3. A method of conducting quality controlto determine abnormalities in operation of a microfluidic system,comprising: detecting a first fluid at a first measurement zone of themicrofluidic system and forming a first signal corresponding to thefirst fluid; transmitting the first signal to a control system;comparing the first signal to a reference signal, thereby determiningthe presence of abnormalities in operation of the microfluidic system;and determining whether to stop an analysis being conducted in themicrofluidic system based at least in part on results of the comparingstep.
 4. A method as in claim 1, comprising continuously or periodicallydetecting the passing of any fluids across the first measurement zone.5. A method as in claim 1, wherein determining whether to modulate fluidflow in the microfluidic system comprises determining whether to stop ananalysis being conducted in the microfluidic system.
 6. A method as inclaim 1, further comprising transmitting an electrical signal from thecontrol system to a component of the microfluidic system that canmodulate fluid flow as a result of the transmitting step.
 7. A method asin claim 1, wherein the component of the microfluidic system is a pumpor a vacuum.
 8. A method as in claim 1, wherein the component of themicrofluidic system is a valve.
 9. A method as in claim 1, furthercomprising comparing the first pattern of signals to a control patternof signals or values pre-programmed into the control system.
 10. Amethod as in claim 1, wherein intensity of the first signal comprises anaverage or maximum intensity.
 11. A method as in claim 1, wherein thefirst pattern of signals comprises an intensity of the first signal anda duration of the first signal.
 12. A method as in claim 1, wherein thefirst pattern of signals comprises an intensity of the first signal anda position of the first signal in time relative to a time of theinitiation step.
 13. A method as in claim 1, wherein the first patternof signals comprises an intensity of the first signal and an averagetime period between the first and second signals.
 14. A method as inclaim 1, further comprising counting a series of signals each having anintensity above or below a threshold intensity, and determining whetherto modulate fluid flow in the microfluidic system based at least in parton the number of signals having the intensity above or below thethreshold intensity.
 15. A method as in claim 1, wherein the first andsecond fluids are immiscible with one another.
 16. A method as in claim1, wherein the first fluid is a liquid and the second fluid is a gas.17. A method as in claim 1, wherein the first and second fluids aremiscible with one another.
 18. A method as in a claim 1, wherein thefirst and second fluids are separated by a third, immiscible fluid. 19.A method as in claim 1, wherein the first fluid is a sample.
 20. Amethod as in claim 1, wherein the first fluid comprises whole blood.21.-37. (canceled)